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J. Neurochem. (2011) 119, 664–672. Abstract Striatal-Enriched protein tyrosine Phosphatase of MW 61 kDa (STEP61) is a protein tyrosine phosphatase recently implicated in the pathophysiology of Alzheimer’s disease (AD). STEP61 is elevated in human AD prefrontal cortex and in the cortex of several AD mouse models. The elevated levels of active STEP61 down-regulate surface expression of GluN1/GluN2B (formerly NR1/NR2B) receptor complexes, while genetically reducing STEP levels rescues both the biochemical and cognitive deficits in a triple transgenic AD mouse model (3xTg-AD). Here, we show that increased STEP61 also plays a role in beta amyloid (Aβ)-mediated internalization of the α-amino-3-hydroxy-5-methyl-4-(AMPA) receptor (AMPAR) subunits GluA1/GluA2 (formerly GluR1/GluR2). We purified Aβ oligomers and determined that oligomers, but not monomers, lead to endocytosis of GluA1/GluA2 receptors in cortical cultures. The decrease in GluA1/GluA2 receptors is reversed in the progeny of STEP knock-out (KO) mice crossed with Tg2576 mice, despite elevated levels of Aβ. These results provide strong support for the hypothesis that STEP61 is required for Aβ-mediated internalization of GluA1/GluA2 receptors.

Protein synthesis underlying activity-dependent synaptic plasticity is controlled at the level of mRNA translation. We examined the dynamics and spatial regulation of two key translation factors, eukaryotic initiation factor 4E (eIF4E) and elongation factor-2 (eEF2), during long-term potentiation (LTP) induced by local infusion of brain-derived neurotrophic factor (BDNF) into the dentate gyrus of anesthetized rats. BDNF-induced LTP led to rapid, transient phosphorylation of eIF4E and eEF2, and enhanced expression of eIF4E protein in dentate gyrus homogenates. Infusion of the extracellular signal-regulated kinase (ERK) inhibitor U0126 blocked BDNF-LTP and modulation of the translation factor activity and expression. Quantitative immunohistochemical analysis revealed enhanced staining of phospho-eIF4E and total eIF4E in dentate granule cells. The in vitro synaptodendrosome preparation was used to isolate the synaptic effects of BDNF in the dentate gyrus. BDNF treatment of synaptodendrosomes elicited rapid, transient phosphorylation of eIF4E paralleled by enhanced expression of alpha-calcium/calmodulin-dependent protein kinase II. In contrast, BDNF had no effect on eEF2 phosphorylation state in synaptodendrosomes. The results demonstrate rapid ERK-dependent regulation of the initiation and elongation steps of protein synthesis during BDNF-LTP in vivo. Furthermore, the results suggest a compartment-specific regulation in which initiation is selectively enhanced by BDNF at synapses, while both initiation and elongation are modulated at non-synaptic sites.

Nicotine, acting on nicotinic acetylcholine receptors (nAChRs) expressed at pre-synaptic dopaminergic terminals, has been shown to stimulate the release of dopamine in the neostriatum. However, the molecular consequences of pre-synaptic nAChR activation in post-synaptic neostriatal neurons are not clearly understood. Here, we investigated the effect of nAChR activation on dopaminergic signaling in medium spiny neurons by measuring phosphorylated DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa) at Thr34 (the PKA-site) in mouse neostriatal slices. Nicotine produced dose-dependent responses, with a low concentration (1 microm) causing a sustained decrease in DARPP-32 Thr34 phosphorylation and a high concentration (100 microm) causing a transient increase in DARPP-32 Thr34 phosphorylation. Depending on the concentration of nicotine, either dopamine D2 or D1 receptor signaling was predominantly activated. Nicotine at a low concentration (1 microm) activated dopamine D2 receptor signaling in striatopallidal/indirect pathway neurons, likely by activating alpha4beta2* nAChRs at dopaminergic terminals. Nicotine at a high concentration (100 microm) activated dopamine D1 receptor signaling in striatonigral/direct pathway neurons, likely by activating (i) alpha4beta2* nAChRs at dopaminergic terminals and (ii) alpha7 nAChRs at glutamatergic terminals, which, by stimulating the release of glutamate, activated NMDA/AMPA receptors at dopaminergic terminals. The differential effects of low and high nicotine concentrations on D2- and D1-dependent signaling pathways in striatal neurons may contribute to dose-dependent actions of this drug of abuse.

Spinophilin is a protein phosphatase-1- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We have recently shown that the interaction of spinophilin with the actin cytoskeleton depends upon phosphorylation by protein kinase A. We have now found that spinophilin is phosphorylated by Ca^(2+)/calmodulin-dependent protein kinase II (CaMKII) in neurons. Ca^(2+)/calmodulin-dependent protein kinase II, located within the post-synaptic density of dendritic spines, is known to play a role in synaptic plasticity and is ideally positioned to regulate spinophilin. Using tryptic phosphopeptide mapping, site-directed mutagenesis and microsequencing analysis, we identified two sites of CaMKII phosphorylation (Ser-100 and Ser-116) within the actin-binding domain of spinophilin. Phosphorylation by CaMKII reduced the affinity of spinophilin for F-actin. In neurons, phosphorylation at Ser-100 by CaMKII was Ca^(2+) dependent and was associated with an enrichment of spinophilin in the synaptic plasma membrane fraction. These results indicate that spinophilin is phosphorylated by multiple kinases in vivo and that differential phosphorylation may target spinophilin to specific locations within dendritic spines.

The striatum is a brain region involved in motor control and in diverse forms of implicit memory. It is also involved in the pathogenesis of many significant human disorders, including drug addiction, that are thought to involve adaptive changes in gene expression. We have previously shown that the cyclin L, ania-6, is expressed as at least two splice forms, which are differentially regulated in striatal neurons by different neurotransmitters. Here, we report that ania-6 transcription is mostly regulated via cAMP response element binding protein (CREB), but that signaling pathways that converge on CREB at the transcriptional level produce different effects on splicing and neuronal gene expression. Glutamate induced a long ania-6 mRNA that encodes a truncated form of the cyclin. This effect depended on the activation of NMDA receptors but was independent of both calcium/calmodulin-dependent protein kinases (CaMK) and extracellular regulated kinase (ERK). Forskolin or brain-derived neurotropic factor (BDNF) induced a short ania-6 mRNA, that encodes the full-length cyclin, and this induction depended on ERK. However, KCl-mediated induction of ania-6 short mRNA, which required activation of L-type calcium channels, was independent of ERK but depended on CaMK. These data suggest that different neuronal signals can differentially regulate splicing and that different intracellular pathways can be recruited to yield a given splice variant.

Methylphenidate (MPH), a dopamine uptake inhibitor, is the most commonly prescribed drug for the treatment of attention-deficit/hyperactivity disorder (ADHD) in children. We examined the effect of MPH on dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32) phosphorylation at Thr34 (PKA-site) and Thr75 (Cdk5-site) using neostriatal slices from young (14-15- and 21-22-day-old) and adult (6-8-week-old) mice. MPH increased DARPP-32 Thr34 phosphorylation and decreased Thr75 phosphorylation in slices from adult mice. The effect of MPH was blocked by a dopamine D1 antagonist, SCH23390. In slices from young mice, MPH did not affect DARPP-32 phosphorylation. As with MPH, cocaine stimulated DARPP-32 Thr34 phosphorylation in slices from adult, but not from young mice. In contrast, a dopamine D1 agonist, SKF81297, regulated DARPP-32 phosphorylation comparably in slices from young and adult mice, as did methamphetamine, a dopamine releaser. The results suggest that dopamine synthesis and the dopamine transporter are functional at dopaminergic terminals in young mice. In contrast, the lack of effect of MPH in young mice is likely attributable to immature development of the machinery that regulates vesicular dopamine release.

The cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator (CFTR), functions as a Cl- channel that is regulated by cyclic AMP-dependent phosphorylation. We have investigated the expression of CFTR protein in the rodent brain by both western blotting of samples prepared by microdissection and immunohistochemistry. CFTR was found to be expressed in choroid plexus and ependyma. In tissue sections, CFTR-like immunoreactivity was concentrated in fine puncta localized about 1-2 microns from the CSF-contacting side of ependyma and choroid plexus. CFTR in choroid plexus may play a role in the regulation of the composition of CSF by cyclic AMP-elevating agents, but the role of this chloride transporter in ependymal function remains to be determined.

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.

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 approximately 25%) the activity of the alpha3 and/or alpha2 isoforms, but not the alpha1 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 alpha3 and/or alpha2 isoforms (by approximately 30%) as well as the alpha1 isoform (by approximately 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 alpha2 or alpha3 isoforms of Na+,K+-ATPase in neostriatal neurons but did increase the phosphorylation of the alpha1 isoform. Thus, in rat neostriatal neurons, treatment with either dopamine or PKC activators results in inhibition of the activity of specific (alpha3 and/or alpha2) 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.

Transient cerebral ischemia, which is accompanied by a sustained release of glutamate and zinc, as well as H(2)O(2) formation during the reperfusion period, strongly depresses protein synthesis. We have previously demonstrated that the glutamate-induced increase in cytosolic Ca(2+) is likely responsible for blockade of the elongation step of protein synthesis, whereas Zn(2+) preferentially inhibits the initiation step. In this study, we provide evidence indicating that H(2)O(2) and thapsigargin mobilized a common intracellular Ca(2+) pool. H(2)O(2) treatment stimulated a slow increase in intracellular Ca(2+), and precluded the effect of thapsigargin on Ca(2+) mobilization. H(2)O(2) stimulated the phosphorylation of both eIF-2alpha and eEF-2, in a time- and dose-dependent manner, suggesting that both the blockade of the elongation and of the initiation step are responsible for the H(2)O(2)-induced inhibition of protein synthesis. However, kinetic data indicated that, at least during the first 15 min of H(2)O(2) treatment, the inhibition of protein synthesis resulted mainly from the phosphorylation of eEF-2. In conclusion, H(2)O(2) inhibits protein translation in cortical neurons by a process that involves the phosphorylation of both eIF-2alpha and eEF-2 and the relative contribution of these two events depends on the duration of H(2)O(2) treatment.

Mice were subjected to 60 min occlusion of the left middle cerebral artery (MCA) followed by 1-6 h of reperfusion. Tissue samples were taken from the MCA territory of both hemispheres to analyse ischaemia-induced changes in the phosphorylation of the initiation factor eIF-2alpha, the elongation factor eEF-2 and p70 S6 kinase by western blot analysis. Tissue sections from additional animals were taken to evaluate ischaemia-induced changes in global protein synthesis by autoradiography and changes in eIF-2alpha phosphorylation by immunohistochemistry. Transient MCA occlusion induced a persistent suppression of protein synthesis. Phosphorylation of eIF-2alpha was slightly increased during ischaemia, it was markedly up-regulated after 1 h of reperfusion and it normalized after 6 h of recirculation despite ongoing suppression of protein synthesis. Similar changes in eIF-2alpha phosphorylation were induced in primary neuronal cell cultures by blocking of endoplasmic reticulum (ER) calcium pump, suggesting that disturbances of ER calcium homeostasis may play a role in ischaemia-induced changes in eIF-2alpha phosphorylation. Dephosphorylation of eIF-2alpha was not paralleled by a rise in levels of p67, a glycoprotein that protects eIF-2alpha from phosphorylation, even in the presence of active eIF-2alpha kinase. Phosphorylation of eEF-2 rose moderately during ischaemia, but returned to control levels after 1 h of reperfusion and declined markedly below control levels after 3 and 6 h of recirculation. In contrast to the only short-lasting phosphorylation of eIF-2a and eEF-2, transient focal ischaemia induced a long-lasting dephosphorylation of p70 S6 kinase. The results suggest that blocking of elongation does not play a major role in suppression of protein synthesis induced by transient focal cerebral ischaemia. Investigating the factors involved in ischaemia-induced suppression of the initiation step of protein synthesis and identifying the underlying mechanisms may help to further elucidate those disturbances directly related to the pathological process triggered by transient cerebral ischaemia and leading to neuronal cell injury.

Ca2+/calmodulin dependent protein kinase (CaMPK) II is a key enzyme in many physiological processes. The enzyme is inactive unless Ca2+/CaM binds to it. In this inactive form CaMPK-II does not bind ATP suggesting that the ATP-binding domain is involved in an intramolecular interaction. We show here that F12, a 12 amino acid long peptide fragment of the ATP-binding domain (CaMPK-II23–34, GAFSVVRRCVKV) can inhibit the Ca2+/CaM-dependent activity (IC50 of 3 µm) but has no effect on the Ca2+/CaM-independent activity of CaMPK-II. Kinetic analysis exhibited mixed inhibition with respect to autocamtide-2 and ATP. The inhibition by F12 showed specificity towards CaMPK-II, but also inhibited CaMPK-I (IC50 = 12.5 µm), while CaMPK-IV (IC50 = 85 µm) was inhibited poorly and cAMP-dependent protein kinase (PKA) was not inhibited. Substitution of phenylalanine at position 25 to alanine (A12), had little effect on the inhibition of different Ca2+/CaM-dependent protein kinases, suggesting that phenylalanine 25 does not play a crucial role in the interactions involving F12. Thus the molecular interactions involving the ATP-binding domain appears to play a role in the regulation of nonphosphorylated CaMPK-II activity.

The Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous protein 1 (WAVE1) is a key regulator of Arp (actin-related protein) 2/3 complex-mediated actin polymerization. We have established previously that the state of phosphorylation of WAVE1 at three distinct residues controls its ability to regulate actin polymerization and spine morphology. Cyclin-dependent kinase 5 phosphorylates WAVE1 at Ser310, Ser397 and Ser441 to a high basal stoichiometry, resulting in inhibition of WAVE1 activity. Our previous and current studies show that WAVE1 can be dephosphorylated at all three sites and thereby activated upon stimulation of the D1 subclass of dopamine receptors and of the NMDA subclass of glutamate receptors, acting through cAMP and Ca(2+) signaling pathways, respectively. Specifically, we have identified protein phosphatase-2A and protein phosphatase-2B as the effectors for these second messengers. These phosphatases act on different sites to mediate receptor-induced signaling pathways, which would lead to activation of WAVE1.

X11-like (X11L) is neuronal adaptor protein that interacts with the amyloid beta-protein precursor (APP) and regulates its metabolism. The phosphotyrosine interaction/binding (PI/PTB) domain of X11L interacts with the cytoplasmic region of APP695. We found that X11L-APP interaction is enhanced in osmotically stressed cells and X11L modification is required for the enhancement. Amino acids 221-250 (X11L(221-250)) are required for the enhanced association with APP in osmotically stressed cells; this motif is 118 amino acids closer to the amino-terminal end of the protein than the PI/PTB domain (amino acids 368-555). We identified two phosphorylatable seryl residues, Ser236 and Ser238, in X11L(221-250) and alanyl substitution of either seryl residue diminished the enhanced association with APP. In brain Ser238 was found to be phosphorylated and phosphorylation of X11L was required for the interaction of X11L and APP. Both seryl residues in X11L(221-250) are conserved in neuronal X11, but not in X11L2, a non-neuronal X11 family member that did not exhibit enhanced APP association in osmotically stressed cells. These findings indicate that the region of X11L that regulates association with APP is located outside of, and amino-terminal to, the PI/PTB domain. Modification of this regulatory region may alter the conformation of the PI/PTB domain to modulate APP binding.

Treatment of PC12 cells with nerve growth factor (NGF), epidermal growth factor (EGF), or agents that raise intracellular cyclic AMP (cAMP) levels (e.g., forskolin) reduces the activity of calmodulin-dependent protein kinase III (CaM-PK III) over a period of 8 h. The mechanism of this effect of NGF has now been examined in more detail, making use of a mutant PC12 cell line (A126-1B2) that is deficient in cAMP-dependent protein kinase activity. Control experiments showed that A 126-1B2 cells retain other NGF-mediated responses (e.g., the induction of ornithine decarboxylase, a cAMP-independent event) and contain a complement of CaM-PK III and its substrate, elongation factor-2, comparable to that of wild-type cells. The ability of NGF or forskolin, but not of EGF, to down-regulate CaM-PK III was markedly attenuated in A 126-1B2 compared to wildtype cells. Treatment of wild-type cells with the cAMP phosphodiesterase inhibitor, isobutylmethylxanthine, enhanced the effects of NGF, but not of EGF. The possibility that NGF led to a stimulation of cAMP-dependent protein kinase activity in wild-type cells was assessed by measurement of the “activation ratio’(-cAMP/+cAMP) of this enzyme before and at various times after NGF addition. A small, but significant, increase in the activation ratio from 0.3 to 0.48 was observed, reaching a peak 5 min after NGF treatment. EGF had no effect on the activation ratio in wild-type cells. These experiments support the hypothesis that NGF, but not EGF, achieves its effects on CaM-PK III by activation of a cAMP-dependent protein kinase and suggest that other actions of NGF may be mediated by the same mechanism.

Cerebral ischaemia is associated with brain damage and inhibition of neuronal protein synthesis. A deficit in neuronal metabolism and altered excitatory amino acid release may both contribute to those phenomena. In the present study, we demonstrate that both NMDA and metabolic impairment by 2-deoxyglucose or inhibitors of mitochondrial respiration inhibit protein synthesis in cortical neurons through the phosphorylation of eukaryotic elongation factor (eEF-2), without any change in phosphorylation of initiation factor eIF-2alpha. eEF-2 kinase may be activated both by Ca(2+)-independent AMP kinase or by an increase in cytosolic Ca2+. Although NMDA decreases ATP levels in neurons, only the effects of 2-deoxyglucose on protein synthesis and phosphorylation of elongation factor eEF-2 were reversed by Na(+) pyruvate. Protein synthesis inhibition by 2-deoxyglucose was not as a result of a secondary release of glutamate from cortical neurons as it was not prevented by the NMDA receptor antagonist 5-methyl-10,11-dihydro-5H-dibenzo-(a,d)-cyclohepten-5,10-imine hydrogen maleate (MK 801), nor to an increase in cytosolic-free Ca2+. Conversely, 2-deoxyglucose likely activates eEF-2 kinase through a process involving phosphorylation by AMP kinase. In conclusion, we provide evidence that protein synthesis can be inhibited by NMDA and metabolic deprivation by two distinct mechanisms involving, respectively, Ca(2+)-dependent and Ca(2+)-independent eEF-2 phosphorylation.

Spinophilin is a protein phosphatase-1 (PP-1)- and actin-binding protein that is enriched in dendritic spines. Phosphorylation of the actin-binding domain of rat spinophilin at one or more sites by protein kinase A (PKA) inhibits actin binding. Here, we investigated the regulation of mouse spinophilin that contains only a single PKA-site (Ser94) within its actin-binding domain. In vitro phosphorylation of Ser94 resulted in the dissociation of spinophilin from actin filaments. In mouse neostriatal slices, phospho-Ser94 (p-Ser94) was dephosphorylated mainly by PP-1 and also by PP-2A. Activation of dopamine D1 receptors in striatonigral medium spiny neurons, and of adenosine A 2A receptors in striatopallidal medium spiny neurons increased, whereas activation of dopamine D2 receptors in striatopallidal neurons decreased, spinophilin Ser94 phosphorylation. In neostriatal slices from DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa) knockout mice, the effects of D1, D2 and A 2A receptors were largely attenuated. Activation of NMDA receptors decreased Ser94 phosphorylation in a PP-2A-dependent, but DARPP-32-independent, manner. These results suggest that PKA-dependent phosphorylation of spinophilin at Ser94 in both striatonigral and striatopallidal neurons requires synergistic contributions from the PKA and DARPP-32/PP-1 pathways. In addition, PP-2A plays a role in Ser94 dephosphorylation in response to activation of both D2 and NMDA receptors.

Methylphenidate (MPH), a dopamine uptake inhibitor, is the most commonly prescribed drug for the treatment of attention-deficit/hyperactivity disorder (ADHD) in children. We examined the effect of MPH on dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32) phosphorylation at Thr34 (PKA-site) and Thr75 (Cdk5-site) using neostriatal slices from young (14-15- and 21-22-day-old) and adult (6-8-week-old) mice. MPH increased DARPP-32 Thr34 phosphorylation and decreased Thr75 phosphorylation in slices from adult mice. The effect of MPH was blocked by a dopamine D1 antagonist, SCH23390. In slices from young mice, MPH did not affect DARPP-32 phosphorylation. As with MPH, cocaine stimulated DARPP-32 Thr34 phosphorylation in slices from adult, but not from young mice. In contrast, a dopamine D1 agonist, SKF81297, regulated DARPP-32 phosphorylation comparably in slices from young and adult mice, as did methamphetamine, a dopamine releaser. The results suggest that dopamine synthesis and the dopamine transporter are functional at dopaminergic terminals in young mice. In contrast, the lack of effect of MPH in young mice is likely attributable to immature development of the machinery that regulates vesicular dopamine release.

The striatum is a brain region involved in motor control and in diverse forms of implicit memory. It is also involved in the pathogenesis of many significant human disorders, including drug addiction, that are thought to involve adaptive changes in gene expression. We have previously shown that the cyclin L, ania-6, is expressed as at least two splice forms, which are differentially regulated in striatal neurons by different neurotransmitters. Here, we report that ania-6 transcription is mostly regulated via cAMP response element binding protein (CREB), but that signaling pathways that converge on CREB at the transcriptional level produce different effects on splicing and neuronal gene expression. Glutamate induced a long ania-6 mRNA that encodes a truncated form of the cyclin. This effect depended on the activation of NMDA receptors but was independent of both calcium/calmodulin-dependent protein kinases (CaMK) and extracellular regulated kinase (ERK). Forskolin or brain-derived neurotropic factor (BDNF) induced a short ania-6 mRNA, that encodes the full-length cyclin, and this induction depended on ERK. However, KCl-mediated induction of ania-6 short mRNA, which required activation of L-type calcium channels, was independent of ERK but depended on CaMK. These data suggest that different neuronal signals can differentially regulate splicing and that different intracellular pathways can be recruited to yield a given splice variant.

ARPP-16 and ARPP-19 are closely related cAMP-regulated phosphoproteins that were initially discovered in mammalian brain as in vitro substrates for protein kinase A (PKA). ARPP-16 is enriched in dopamine-responsive medium spiny neurons in the striatum, while ARPP-19 is ubiquitously expressed. ARPP-19 is highly homologous to alpha-endosulfine and database searches allowed the identification of novel related proteins in D. melanogaster, C. elegans, S. mansoni and yeast genomes. Using isoform-specific antibodies, we now show that ARPP-19 is composed of at least two differentially expressed isoforms (termed ARPP-19 and ARPP-19e/endosulfine). All ARPP-16/19 family members contain a conserved consensus site for phosphorylation by PKA (RKPSLVA in mammalian ARPP-16 and ARPP-19), and this site was shown to be efficiently phosphorylated in vitro by PKA. An antibody that specifically recognized the phosphorylated form of ARPP-16/19/19e was used to examine the phosphorylation of ARPP-16/19 family members in intact cells. In striatal slices, the phosphorylation of ARPP-16 was increased in response to activation of D(1)-type dopamine receptors, and decreased in response to activation of D(2)-type dopamine receptors. In non-neuronal cells, ARPP-19 was highly phosphorylated in response to activation of PKA. These results establish that ARPP-16/19 proteins constitute a family of PKA-dependent intracellular messengers that function in all cells. The high levels of ARPP-16 in striatal neurons and its bi-directional regulation by dopamine suggest a specific role in dopamine-dependent signal transduction. The conservation of this protein family through evolution suggests that it subserves an important cellular function that is regulated by PKA.

The mature form of Alzheimer's β-amyloid precursor protein (APP) is phosphorylated specifically at Thr668 in neurons. In mature neurons, phosphorylated APP is detected in neurites, with dephosphorylated APP being found mostly in the cell body. In vitro, active cyclin-dependent kinase 5 (Cdk5) phosphorylated the cytoplasmic domain of APP at Thr668. Treatment of mature neurons with an antisense oligonucleotide to Cdk5 suppressed Cdk5 expression and significantly diminished the level of phosphorylated APP. The expression of APP was unaffected in antisense-treated neurons. These results indicate that in neurons APP is phosphorylated by Cdk5, and that this may play a role in its localization.

Alzheimer amyloid precursor protein (APP) is an integral membrane protein with a short cytoplasmic domain of 47 amino acids. It is hoped that identification of proteins that interact with the cytoplasmic domain will provide new insights into the physiological function of APP and, in turn, into the pathogenesis of Alzheimer's disease. To identify proteins that interact with the cytoplasmic domain of APP, we employed affinity chromatography using an immobilized synthetic peptide corresponding to residues 645-694 of APP695 and identified a protein of approximately 130 kDa in rat brain cytosol. Amino acid sequencing of the protein revealed the protein to be a rat homologue of monkey UV-DDB (UV-damaged DNA-binding protein, calculated molecular mass of 127 kDa). UV-DDB/p127 co-immunoprecipitated with APP using an anti-APP antibody from PC12 cell lysates. APP also co-immunoprecipitated with UV-DDB/p127 using an anti-UV-DDB/p127 antibody. These results indicate that UV-DDB/p127, which is present in the cytosolic fraction, forms a complex with APP through its cytoplasmic domain. In vitro binding experiments using a glutathione S-transferase-APP cytoplasmic domain fusion protein and several mutants indicated that the YENPTY motif within the APP cytoplasmic domain, which is important in the internalization of APP and amyloid beta protein secretion, may be involved in the interaction between UV-DDB/p127 and APP.

The distribution of inhibitor-1, a cyclic AMP-regulated inhibitor of protein phosphatase-1, was analyzed in various brain regions and peripheral tissues of various species by immunolabeling of sodium dodecyl sulfate-poly-acrylamide gel transfers using specific antibodies. The distribution of inhibitor-1 was directly compared to that of DARPP-32, a structurally related cyclic AMP-regulated inhibitor of protein phosphatase-1. In rat CNS, a single immunoreactive protein of M(r) 30,000, identified as inhibitor-1, was widely distributed. In contrast, DARPP-32 was highly concentrated in the basal ganglia. Inhibitor-1 was detected in brain tissue from frog (M(r) 27,000), turtle (M(r) 29,000/33,000), canary (M(r) 26,000), pigeon (M(r) 28,000), mouse (M(r) 30,500), rabbit (M(r) 26,500), cow (M(r) 27,000), and monkey (M(r) 27,500), but not from goldfish. Inhibitor-1 was detected at various levels in most peripheral tissues of the species studied; however, it was not detectable in certain tissues of particular species (e.g., rat and cow liver). DARPP-32 was detected in brain tissue of all the species tested except frog and goldfish, but was not detectable in most peripheral tissues. Both inhibitor-1 and DARPP-32 were concentrated in the cytosol and synaptosomal cytosol of rat striatum. The developmental expressions of inhibitor-1 and DARPP-32 in rat striatum differed: the level of inhibitor-1 peaked in the first postnatal week and then declined by the third postnatal week, whereas the level of DARPP-32 increased to a peak level by the third postnatal week and remained elevated thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)