Synapsins are neuronal phosphoproteins that coat synaptic vesicles, bind to the cytoskeleton, and are believed to function in the regulation of neurotransmitter release. Molecular cloning reveals that the synapsins comprise a family of four homologous proteins whose messenger RNA's are generated by differential splicing of transcripts from two genes. Each synapsin is a mosaic composed of homologous amino-terminal domains common to all synapsins and different combinations of distinct carboxyl-terminal domains. Immunocytochemical studies demonstrate that all four synapsins are widely distributed in nerve terminals, but that their relative amounts vary among different kinds of synapses. The structural diversity and differential distribution of the four synapsins suggest common and different roles of each in the integration of distinct signal transduction pathways that modulate neurotransmitter release in various types of neurons.

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 diseasecausing 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, Mr 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 D1 dopamine͞DARPP-32 signaling cascade. These presymptomatic defects may contribute to HD pathology. H untington's disease (HD) is an inherited neurodegenerative disorder characterized by progressive motor, psychiatric, and cognitive disturbances. The motor disturbance begins subtly, as minor adventitious movements, and gradually progresses until the entire body is consumed in flagrant chorea and uncontrolled writhing, leading to death 10-20 years after onset (1). HD pathology is characterized by specific neurodegeneration of the caudate nucleus and putamen (2, 3). Genetic analysis has established a causative link between HD and abnormal expansion of a polyglutamine repeat in the protein huntingtin (4). The transgenic mouse line R6͞2 (HD mice) is a well-characterized animal model of HD in which a fragment of a disease-causing variant of human huntingtin is ectopically expressed. These mice exhibit HD-like behavioral changes beginning after 8 weeks of life, progressively deteriorate over the succeeding 10 weeks, and die prematurely (5, 6). Although the HD mice are essentially asymptomatic until week 8, behavioral deficits are preceded by the appearance of intranuclear inclusion bodies in the central nervous system (7,8). These inclusions are similar to those detected in HD and other diseases associated with polyglutamine expansions (7-9).Both normal and mutant huntingtin are expressed throughout the body. The dramatic and selective loss of striatal tissue seen in HD, and the resultant pronounced behavioral and pathological effects, have remained unexplained (1, 10, 11). Given that the striatum is the predominant target of midbrain dopamine neurons, we sought to determine whether dopamine signaling might be altered in presymptomatic HD mice. We found that while many parameters of neuronal function remained unchanged, there were significant deficits including severely impaired electrophysiological and biochemical responses to activation of D1-class dopamine receptors, which precede the emergence of histopathological and behavioral changes. Materials and MethodsForskolin was purchased from LC Laboratories. 8-BromocAMP and protease type XIV were purchased from Sigma. SKF81297, D1 receptor antibodies, kainic acid, dopamine, and ␥-aminobutyric acid (GABA) were purchased from Research Biochemicals. Actin antibodies were purchased from BRL....

Phospho-DARPP-32 (where DARPP-32 is dopamineand cAMP-regulated phosphoprotein, M r 32,000), its homolog, phospho-inhibitor-1, and inhibitor-2 are potent inhibitors (IC 50 ϳ1 nM) of the catalytic subunit of protein phosphatase-1 (PP1). Our previous studies have indicated that a region encompassing residues 6 -11 (RKKIQF) and phospho-Thr-34, of phospho-DARPP-32, interacts with PP1. However, little is known about specific regions of inhibitor-2 that interact with PP1. We have now characterized in detail the interaction of phospho-DARPP-32 and inhibitor-2 with PP1. Mutagenesis studies indicate that within DARPP-32 Phe-11 and Ile-9 play critical roles, with Lys-7 playing a lesser role in inhibition of PP1. Pro-33 and Pro-35 are also important, as is the number of amino acids between residues 7 and 11 and phospho-Thr-34. For inhibitor-2, deletion of amino acids 1-8 (I2-(9 -204)) or 100 -204 (I2-(1-99)) had little effect on the ability of the mutant proteins to inhibit PP1. Further deletion of residues 9 -13 (I2-(14 -204)) resulted in a large decrease in inhibitory potency (IC 50 ϳ800 nM), whereas further COOH-terminal deletion (I2-(1-84)) caused a moderate decrease in inhibitory potency (IC 50 ϳ10 nM). Within residues 9 -13 (PIKGI), mutagenesis indicated that Ile-10, Lys-11, and Ile-13 play critical roles. The peptide I2-(6 -20) antagonized the inhibition of PP-1 by inhibitor-2 but had no effect on inhibition by phospho-DARPP-32. In contrast, the peptide D32-(6 -38) antagonized the inhibition of PP1 by phospho-DARPP-32, inhibitor-2, and I2-(1-120) but not I2-(85-204). These results indicate that distinct amino acid motifs contained within the NH 2 termini of phospho-DARPP-32 (KKIQF, where italics indicate important residues) and inhibitor-2 (IKGI) are critical for inhibition of PP1. Moreover, residues 14 -84 of inhibitor-2 and residues 6 -38 of phospho-DARPP-32 share elements that are important for interaction with PP1.Protein phosphatase-1 (PP1) 1 is a major eukaryotic protein serine/threonine phosphatase that regulates diverse cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription, and neuronal signaling (1-4). The catalytic subunit of PP1 is regulated by the heat-stable protein inhibitors, inhibitor-1, its homolog DARPP-32 (dopamine-and cAMP-regulated phosphoprotein, M r 32,000), and inhibitor-2 (1, 2). Phosphorylation of inhibitor-1 at Thr-35 or of DARPP-32 at Thr-34 by cAMP-dependent protein kinase converts either protein into a potent inhibitor of PP1. In contrast, unphosphorylated inhibitor-2 interacts with the catalytic subunit of PP1 leading first to inhibition of enzyme activity and subsequently to an inactive complex, termed Mg-ATP-dependent PP1 (1, 5). The Mg-ATPdependent form of PP1 can then be re-activated following phosphorylation of Thr-72 of inhibitor-2 by glycogen synthase kinase-3 (GSK-3). PP1 is also regulated by its interaction with a variety of protein subunits that act in a manner distinct from the inhibitor proteins and t...

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 glutamatedependent regulation of N-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.Control of protein phosphorylation/dephosphorylation occurs through regulation of protein kinase and protein phosphatase activities and is an integral component of intracellular signal transduction. Inhibitor-1 was the first endogenous molecule found to regulate protein phosphatase activity (1). Inhibitor-1 purified from rabbit skeletal muscle is an 18,700-kDa acid-and heat-stable protein composed of 166 amino acids that are highly conserved throughout phylogeny (2, 3). When phosphorylated at Thr-35 by cAMP-dependent protein kinase (PKA), 1 inhibitor-1 selectively and potently inhibits type 1 protein phosphatase (protein phosphatase-1, PP-1) with an IC 50 value of ϳ1 nM (4 -7). Phospho-Thr-35 inhibitor-1 is dephosphorylated by Ca 2ϩ /calmodulin-dependent protein phosphatase 2B (PP-2B, calcineurin) and protein phosphatase 2A (PP-2A), with PP-2B activity predominating in the presence of Ca 2ϩ (8 -11). First messengers such as neurotransmitters (e.g. dopamine and acetylcholine) and hormones (e.g. adrenaline) that elevate intracellular cAMP levels promote PKA-dependent phosphorylation of inhibitor-1 at Thr-35 in various tissues. PP-1 inhibition by phospho-Thr-35 inhibitor-1 provides substantial amplification of PKA-dependent signaling cascades and modulates the intensity and duration of a number of physiological responses including regulatory aspects of the cell cycle, gene expression, carbohydrate and lipid metabolism, and synaptic plasticity (12-17).Inhibitor-1 is widely expressed in mammalian tissue with highest levels occurring in the brain, skeletal muscle, adipose, and kidney tissues (18 -26). Within the brain, the highest levels of inhibitor-1 i...

Site-directed mutagenesis of selected residues of mammalian protein phosphatase 1 (PP-1) has been carried out to further define the mechanism of catalysis, activation by divalent cations, and inhibition by toxins and inhibitory proteins. Mutation of active site residues predicted to bind metals (N124D and H248N) resulted in a large loss of enzyme activity and decreased affinity for metal ions; mutation of residues predicted to bind phosphosubstrate (R96A or R221S) led to a large loss of enzyme activity; and mutation of active site residues (D95A and D208A) resulted in a large loss of enzyme activity. Mutants N124D, H248N, R96A, and R221S exhibited large decreases in sensitivity to the toxins calyculin A, okadaic acid, and microcystin and to thiophospho-DARPP-32. Mutation of Y272 (Y272F) had little effect on activity but resulted in a large decrease in sensitivity to okadaic acid and calyculin A. Mutant D208A exhibited a decrease in sensitivity to okadaic acid and calyculin A, but, paradoxically, the sensitivity to inhibition by thiophospho-DARPP-32 was increased. Mutation of acidic groove residues (E256R, E275R, E252A:D253A, and E252A:D253A:E256R) exhibited little change in enzyme activity and no change in sensitivity to toxins, but increased sensitivity to thiophospho-DARPP-32. These results suggest that toxins and phospho-DARPP-32 interact at the active site of PP-1 in a similar fashion despite their differences in structure. In addition, acidic groove residues appear to inf luence the interaction of the phosphoinhibitor with the active site of PP-1.Based on their biochemical properties, in particular substrate specificity and sensitivity to divalent cations and protein inhibitors, serine͞threonine protein phosphatases (PPases) have been classified into four major types (PP-1, -2A, -2B, and -2C). The catalytic subunits of PP-1, -2A, and -2B (referred to here as the PPase family) show a high degree of sequence identity (40-50%) within a region of Ϸ30 kDa, suggesting that these enzymes share a conserved structure and mechanism of catalysis (1-3). In contrast, the amino acid sequence of PP-2C is unrelated to any of the PPase family members and is likely to have a distinct structure and enzyme mechanism. The catalytic subunits of the PPases are subject to regulation by a variety of interacting subunits, targeting proteins and inhibitors (1, 3-5). For example, PP-1 is regulated by the heat-stable proteins, inhibitor-1, the related homolog DARPP-32 (dopamine and cAMP-regulated phosphoprotein of M r 32,000), and inhibitor-2. Phosphorylation of inhibitor-1 (at Thr-35) or DARPP-32 (at Thr-34) by cAMP-dependent protein kinase (PKA) converts them into potent inhibitors of PP-1. PP-1 and PP-2A, but not PP-2B, are also highly sensitive to inhibition by a number of tumor-promoting natural toxins, for example, okadaic acid, calyculin A, and microcystin (3). In addition, PP-1 is inhibited by phosphorylation of a threonine residue (Thr-320) in the COOH-terminal domain of the protein by cyclin-dependent protein kinase (...