Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.16 (calcineurin)
 17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000) is a major endogenous cytosolic substrate for dopamine- and cyclic AMP-stimulated protein phosphorylation in neurons of the basal ganglia of mammalian brain. It shares many properties with phosphatase inhibitor 1, a substrate for cyclic AMP-dependent protein kinase, and with G-substrate, a substrate for cyclic GMP-dependent protein kinase. We have, therefore, undertaken an analysis of the amino acid sequence around the site at which purified DARPP-32 is phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. The results indicate that DARPP-32 is phosphorylated at a single threonine residue contained in the sequence Arg-Arg-Arg-Pro-Thr(P)-Pro-Ala-Met-Leu-Phe-Arg. This sequence was obtained by automated solid phase sequencing of two overlapping tryptic phosphopeptides and one overlapping chymotryptic phosphopeptide which were purified by reverse-phase high-performance liquid chromatography. A 9-amino acid sequence containing the phosphorylatable threonine residue in DARPP-32 shares 8 identical residues with a sequence containing the phosphorylatable threonine residue in phosphatase inhibitor 1, and shares 5 identical residues with the two identical sequences surrounding the 2 phosphorylatable threonine residues in G-substrate. These observations support the view that DARPP-32, inhibitor 1, and G-substrate are members of a family of regulatory proteins which are involved in the control of protein phosphatase activity by both cyclic AMP and cyclic GMP, but which differ in their cellular and tissue distributions.
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PMID:DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated neuronal phosphoprotein. I. Amino acid sequence around the phosphorylated threonine. 650 2

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000) is a cytosolic neuronal phosphoprotein enriched in dopamine-innervated brain regions which, in its phosphorylated form, acts as an inhibitor of protein phosphatase 1. We have compared the phosphorylation of purified DARPP-32 with that of purified phosphatase inhibitor 1, a widespread inhibitor of protein phosphatase 1. Purified cyclic AMP-dependent protein kinase and cyclic GMP-dependent protein kinase each catalyzed the maximal incorporation of 0.9-1.1 mol of [32P]phosphate/mol of DARPP-32 or phosphatase inhibitor 1, with phosphorylation occurring on threonine residues. Evidence for the existence of a single phosphorylation site in each substrate protein was obtained by two-dimensional thin-layer phosphopeptide mapping of thermolytic digests. Initial rate studies of the phosphorylation of DARPP-32 yielded an apparent Km of 2.4 microM and a kcat of 2.7 S-1 for the catalytic subunit of cyclic AMP-dependent protein kinase, and an apparent Km of 5.4 microM and a kcat of 2.3 S-1 for cyclic GMP-dependent protein kinase. These in vitro results are compatible with a physiological role for the phosphorylation of DARPP-32 by either protein kinase in vivo. Similar studies with phosphatase inhibitor 1 yielded an apparent Km of 5.0 microM and a kcat of 1.4 S-1 for the catalytic subunit of cyclic AMP-dependent protein kinase, and an apparent Km of 25.0 microM and a kcat of 1.2 S-1 for cyclic GMP-dependent protein kinase. A synthetic nonapeptide, corresponding to the phosphorylation site of DARPP-32, was phosphorylated with apparent Km values of 1.12 mM and 1.86 mM and kcat values of 0.22 S-1 and 3.4 S-1 for cyclic AMP-dependent and cyclic GMP-dependent protein kinase, respectively.
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PMID:DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated neuronal phosphoprotein. II. Comparison of the kinetics of phosphorylation of DARPP-32 and phosphatase inhibitor 1. 650 3

DARPP-32, a dopamine- and adenosine 3',5'-cyclic monophosphate (cAMP)-regulated inhibitor of protein phosphatase-1, is highly colocalized with neuronal and nonneuronal D1-type receptors. DARPP-32 concentration is enriched in the renal outer medulla and in the medium-size spiny neurons of the brain. In the ascending limb of the loop of Henle, DARPP-32 is phosphorylated following stimulation by dopamine and other first messengers, and in this form inhibits the activity of the Na(+)-K(+)-adenosinetriphosphatase pump. For functional analysis of the DARPP-32 promoter in the kidney, we characterized the murine gene. There are two groups of transcription start sites utilized in the brain, but the proximal set appears to be preferentially used in the kidney. In four of four lines of mice carrying a DARPP-32/lacZ transgene with 2.1 kb of 5'-flanking DNA, adult kidney lacZ transgene expression mimicked that of endogenous DARPP-32. There was no ectopic expression in peripheral organs. We conclude that the sequences necessary for direction of DARPP-32 expression to the medullary thick ascending limb are contained within this 2.1-kb fragment.
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PMID:DARPP-32 promoter directs transgene expression to renal thick ascending limb of loop of Henle. 748 43

Previous studies of hormonal regulation of renal Na(+)-K(+)-ATPase have indicated that the activity of the sodium pump is regulated by phosphorylation-dephosphorylation reactions. Here we report that okadaic acid (OA) and calyculin A (CL-A), inhibitors of protein phosphatase (PP)-1 and PP-2A, inhibited Na(+)-K(+)-ATPase activity in cells from the rat thick ascending limb (TAL) of loop of Henle in a dose-dependent manner. CL-A was 10-fold more potent than OA. On the basis of the inhibitory constant values of CL-A and OA for PP-1 and PP-2A, it is concluded that the tubular effect is mainly due to inhibition of PP-1. In situ hybridization studies with oligonucleotide probes revealed very strong PP-1 alpha and PP-1 gamma 1 mRNA labeling in the outer stripe of the outer medulla, strong labeling in the inner stripe of the outer medulla, and weak labeling in the inner medulla. Very weak labeling was demonstrated in the outer cortex. PP-1 beta mRNA labeling was very strong in the inner stripe of the outer medulla, whereas the outer stripe had weaker labeling, and the inner medulla had weak labeling. PP-1 alpha, PP-1 beta, and PP-1 gamma 1 mRNA were also demonstrated in the transitional epithelium of the ureter. The abundance of the PP-1 alpha and PP-1 gamma isoforms as measured by immunoblotting was very high in tissue from the outer medulla, which also has a high abundance of the endogenous dopamine-regulated PP-1 inhibitor, DARPP-32.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Protein phosphatase-1 in the kidney: evidence for a role in the regulation of medullary Na(+)-K(+)-ATPase. 750 33

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.
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PMID:Dopamine- and cAMP-regulated phosphoprotein DARPP-32: phosphorylation of Ser-137 by casein kinase I inhibits dephosphorylation of Thr-34 by calcineurin. 770 5

DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000) is a potent inhibitor of protein phosphatase-1 when it is phosphorylated on Thr-34 by cAMP-dependent protein kinase. DARPP-32 is highly enriched in some specific cell populations such as striatonigral neurons and choroid plexus epithelial cells. Here we show that recombinant rat DARPP-32 is phosphorylated by casein kinase I on seryl residues to a stoichiometry of approximately 2 mol of phosphate/mol of protein. DARPP-32 is one of the best known substrates for casein kinase I (Km = 3.4 +/- 0.3 microM), whereas the homologous phosphatase-1 inhibitor, inhibitor-1, is not. Phosphorylation of DARPP-32 by casein kinase I does not alter its ability to inhibit protein phosphatase-1. Residues phosphorylated by casein kinase I were identified as Ser-137 and Ser-189 by site-directed mutagenesis and by protein sequencing. Ser-137 and the preceding stretch of 16-18 acidic residues are conserved in DARPP-32 among all species examined, whereas Ser-189 is not. Phosphorylation of Ser-137 induces an unusual increase in DARPP-32 electrophoretic mobility in polyacrylamide gels in the presence of SDS. In striatonigral neurons, DARPP-32 is phosphorylated on Ser-137 and the stoichiometry of phosphorylation on this residue in vivo appears to be higher in the substantia nigra (axon terminals) than in the striatum (soma and dendrites). These results indicate that casein kinase I is highly active in striatonigral neurons in which it may play important roles, including in protein phosphatase-1 modulation via phosphorylation of DARPP-32.
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PMID:Phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by casein kinase I in vitro and in vivo. 772 83

A distinct form of DARPP-32, a protein phosphatase-1 inhibitor, has been identified in bovine calf parathyroid glands. Immunoblot analysis of parathyroid tissue revealed a 32 kDa protein present predominantly in a particulate fraction; it remained particulate after treatment with 1.0 M NaCl or 0.1 M Na2CO3. Metabolic labeling of parathyroid cells with mevalonolactone demonstrated that DARPP-32 is isoprenylated. Immunocytochemical localization studies demonstrated that DARPP-32 is present in vesicles throughout the cytoplasm of parathyroid cells, and that protein phosphatase-1 gamma is concentrated in the region of the plasma membrane. Thus, in contrast to the predominately soluble form of DARPP-32 that has been characterized in selected areas of the central nervous system, the parathyroid form is tightly associated with intracellular membranes.
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PMID:DARPP-32 (dopamine and cAMP-regulated phosphoprotein, M(r) 32,000) is a membrane protein in the bovine parathyroid. 775 May 46

Rat cDNAs encoding neuronal isoforms of protein phosphatase 1 (PP1) were isolated and their primary structures elucidated. The derived amino acid sequences allowed us to design synthetic C-terminal peptides that were used to raise antibodies. Isoform-specific anti-peptide antibodies against PP1 alpha and PP1 gamma 1 were used to investigate the tissue distribution of PP1 isoforms by immunoblotting. Both isoforms were ubiquitously expressed in mammalian tissues, with the highest levels being observed in brain. Of all neuronal tissues examined, PP1 alpha and PP1 gamma 1 were found to be most abundantly expressed in the striatum. Lesion experiments with kainic acid indicated that both the alpha and the gamma 1 isoforms of protein phosphatase 1 were relatively enriched in the medium-size spiny neurons of the striatum. "In situ" hybridization to rat brain slices using highly sensitive riboprobes also showed PP1 alpha, PP1 beta, and PP1 gamma 1 to be widely expressed in mammalian brain. However, some interesting differences were observed. For example, PP1 alpha and PP1 gamma 1 were found to be expressed in the striatum, where DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000 Da) is also known to be highly expressed. PP1 beta appeared to be relatively less abundant in the same cells, as judged both by "in situ" hybridization and by the apparent absence of PP1 beta clones from the striatal cDNA libraries used.
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PMID:Differential expression of protein phosphatase 1 isoforms in mammalian brain. 775 17

It is hypothesized that the immunosuppressive agents cyclosporin A and FK-506 may elicit a dopamine-like effect upon dopaminoceptive neurons in the striatum. When complexed to their immunophilins, these molecules will inhibit calcineurin activity leading to increased phosphorylation of dopamine- and cAMP-regulated phosphoprotein (DARPP-32) and hence, inhibition of protein phosphatase-1 activity. As a net result, intracellular protein phosphorylation increases. One or more of these proteins may, in their phosphorylated form, inhibit the depolarization of the neurons, resulting in a dopamine-like effect.
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PMID:Calcineurin as a possible new target for treatment of Parkinson's disease. 781 62

The mechanism of inhibition of protein phosphatase-1 catalytic subunit (PP-1c) by recombinant DARPP-32 and synthetic peptides was studied. DARPP-32 was expressed in Escherichia coli as a non-fusion protein using a pEt-3a plasmid, purified to homogeneity and shown to have physicochemical properties similar to those of the protein purified from bovine brain. Recombinant DARPP-32 phosphorylated on threonine-34 by cAMP-dependent protein kinase inhibited PP-1c with an IC50 approximately 0.5 nM, comparable to that obtained with bovine DARPP-32. Non-phosphorylated DARPP-32, and mutated forms in which threonine-34 was replaced by an alanine or a glutamic acid, inhibited PP-1c with an IC50 approximately 1 microM. Surface plasmon resonance analysis showed binding of PP-1c to nonphospho- and phospho-DARPP-32-(8-38) synthetic peptides with apparent Kd values of 1.2 and 0.3 microM, respectively, supporting the existence of an interaction between non-phosphorylated DARPP-32 and PP-1c that is increased by phosphorylation of DARPP-32 at threonine-34. These results suggest a model in which DARPP-32 interacts with PP-1c by at least two low affinity sites, the combination of which is responsible for the high affinity (nM) inhibition.
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PMID:Mechanism of inhibition of protein phosphatase 1 by DARPP-32: studies with recombinant DARPP-32 and synthetic peptides. 782 84


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