Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

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

In the brain, dopamine, via protein kinase A (PKA) activation of dopamine- and cAMP-regulated phosphoprotein (DARPP-32), inhibits protein phosphatase 1 (PP1) activity and keeps Na(+)-K(+)-adenosinetriphosphatase (ATPase) in its phosphorylated inactive state. In the present study, we examined the relationship among dopamine, PP1, and Na(+)-K(+)-ATPase activities in renal proximal tubules. PP1 activity in proximal tubules was not decreased by dopamine (5 x 10(-9)-10(-4) M), fenoldopam (5 x 10(-6) M), or norepinephrine (5 x 10(-7) M). In contrast, in the medullary thick ascending limb of Henle and in the brain striatum, PP1 activity was decreased by fenoldopam (5 x 10(-6) M). We also showed that the ability of dopamine (10(-6) M) to inhibit Na(+)-K(+)-ATPase activity in proximal tubules (assessed by ouabain-sensitive 86Rb uptake) occurred in the absence or presence of a sodium clamp with 5 microM monensin. Thus the inhibitory effect of dopamine on Na(+)-K(+)-ATPase activity in proximal tubules is not regulated by PP1 activity. Tautomycin and okadaic acid by themselves, at concentrations that inhibited PP1 activity, had no effect on Na(+)-K(+)-ATPase activity in proximal tubules. The ability of a dopamine D1 agonist, fenoldopam, to inhibit PP1 activity in brain striatum and in medullary thick ascending limb, but not in proximal tubules, suggests differential organ and nephron segment regulation of PP activity.
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PMID:Dopamine and protein phosphatase activity in renal proximal tubules. 786 67

In the medium-sized spiny neurons of the striatonigral pathway, a cascade of events involving the activation of dopamine D1 receptors, an increase in cyclic AMP, and activation of cyclic AMP-dependent protein kinase causes the phosphorylation of DARPP-32 on Thr34, converting DARPP-32 into a powerful inhibitor of protein phosphatase-1. In the present study, the incubation of striatal or substantia nigra slices with GABA also increased the phosphorylation of DARPP-32 on Thr34. GABA did not significantly increase cyclic AMP levels in slices. The phosphorylation of DARPP-32 by GABA was blocked in both brain regions by pretreatment of slices with the GABAA receptor antagonist, bicuculline, but not with the GABAB receptor antagonist, phaclofen. Moreover, the threonine phosphorylation of DARPP-32 produced by maximally effective doses of either forskolin (in striatum) or L-3,4-dihydroxyphenylalanine (in substantia nigra) was increased further by GABA. The data are consistent with a model in which GABA increases the phosphorylation state of DARPP-32 by inhibiting dephosphorylation of the protein by the calcium/calmodulin-dependent protein phosphatase, calcineurin.
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PMID:Phosphorylation of DARPP-32 is regulated by GABA in rat striatum and substantia nigra. 793 32

The clinical efficacy of dopamine (DA) replacement therapy for patients with Parkinson's disease (PD) depends on the preservation of postsynaptic DA receptors and their intracellular signalling mechanisms in the striatum long after degeneration of the nigrostriatal DA pathway. DA activates adenylyl cyclase (AC) and phospholipase C (PLC) via the D1 receptor, and inhibits through the D2 receptor, thereby regulating the production of intracellular second messengers, cyclic adenosine 3',5'-monophosphate (cAMP), 1,2-diacylglycerol (DAG) and Ca2+. Recent advances in molecular biology have made it possible to monitor the intracellular signal transduction cascade following receptor activation by various transmitters. The authors review the literature addressing this issue, summarized as follows: (1) striatal D1 and D2 receptor densities remain constant, at least in treated and non-demented patients; (2) DA-sensitive AC activity appears to be increased in the putamen of treated patients, although this remains to be confirmed; (3) levels of cAMP-dependent protein kinase (PKA) are normal in non-demented patients, consistent with unchanged levels of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of M(r) 32,000); (4) levels of Ca2+/phospholipid-dependent protein kinase (PKC) and of inositol 1,4,5-trisphosphate (InsP3) receptor also remain unchanged in non-demented patients; (5) the above three second messenger sites as well as densities of D1 and D2 receptors are decreased in the striatum of demented PD patients (PDD). We tentatively conclude that postreceptor signalling function is intact in the striatum of non-demented PD patients and that there is a clear difference between non-demented patients and PDD, i.e. striatal dopaminoceptive neurons are affected in PDD.
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PMID:Transmembrane signalling systems in the brain of patients with Parkinson's disease. 795 88

DARPP-32 is a potent inhibitor of protein phosphatase 1 when it is phosphorylated on Thr34 by cAMP-dependent protein kinase. DARPP-32 is also phosphorylated on Ser45 and Ser102 by casein kinase II, resulting in a facilitation of phosphorylation by cAMP-dependent protein kinase. We have studied the conformation of recombinant rat DARPP-32 by steady-state and time-resolved fluorescence. The steady-state emission spectra and quenching of the intrinsic (Trp163) and extrinsic fluorescence (acrylodan or lucifer yellow linked to Cys72) were consistent with a complete exposure of these residues to the aqueous environment. The intrinsic fluorescence of DARPP-32 was resolved into three decay components with lifetimes of 1, 3.4, and 7 ns, with the intermediate lifetime component giving the major contribution. The ratio between the amplitudes associated with the short and long decay constants was decreased upon denaturation. The rotational behavior of DARPP-32 measured by anisotropy decay revealed that Trp163 is located in a highly flexible peptide chain, whereas Cys72 is embedded in a more rigid environment. Phosphorylation by cAMP-dependent protein kinase did not alter any of the fluorescence parameters, whereas only minor effects were associated with casein kinase II phosphorylation. These findings indicate that DARPP-32 contains at least two distinct domains and that phosphorylation has no dramatic effects on its conformation.
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PMID:Study of the conformation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by fluorescence spectroscopy. 822 46

DARPP-32, a dopamine- and cAMP-regulated phosphoprotein of M(r) 32,000, has been shown to be phosphorylated on threonine-34, both in vitro with high efficiency by cAMP-dependent and cGMP-dependent protein kinases and in vivo by dopamine acting through cAMP-dependent protein kinase. In the present study, we investigated the nitric oxide (NO)/cGMP pathway for its ability to regulate the state of phosphorylation of DARPP-32 in slices of rat substantia nigra. DARPP-32 was phosphorylated on threonine-34 in these slices by sodium nitroprusside (SNP), an NO donor. The effect of SNP was abolished by preincubation of the slices with hemoglobin, indicating that the effect of SNP was due to released NO. The same concentration of SNP produced a 4-fold elevation of the cGMP level but did not alter the level of cAMP. The effect of SNP on DARPP-32 phosphorylation was mimicked by low concentrations of 8-bromo-cGMP and 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate, activators of cGMP-dependent protein kinase, but not by low concentrations of 8-bromo-cAMP, an activator of cAMP-dependent protein kinase. The data indicate a physiological role for the NO/cGMP pathway in the regulation of DARPP-32 phosphorylation in nerve terminals of striatonigral neurons. The results provide further evidence that the state of phosphorylation of DARPP-32 represents an important mechanisms for integration of information arriving at striatonigral neurons via a variety of neuronal pathways.
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PMID:Nitric oxide/cGMP pathway stimulates phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, in the substantia nigra. 838 74

Immotile bovine caput epididymal sperm contain levels of protein phosphatase activity twofold higher than do mature motile caudal sperm. Comparison of the inhibition profiles of endogenous phosphatase activities detected by okadaic acid (OA) and calyculin A (CA) revealed a pattern consistent with the predominance of a type 1 protein phosphatase (PP1). Immunoblot analysis identified PP1 gamma 2 (the testis-specific isoform of PP1) as the only PP1 isoform in sperm and showed little protein phosphatase 2A (PP2A). In addition, of the known PP1 inhibitors, i.e., DARPP-32, inhibitor 1 (I1), and inhibitor 2 (I2), only I2-like activity was detected in sperm. Inhibition of PP1 by the heat-stable I2-like activity purified from sperm could be reversed with purified glycogen synthase kinase-3 (GSK-3). Furthermore, sperm extracts contain an inactive complex of PP1 and I2 (termed PP1I) that could also be activated by purified GSK-3. The presence of GSK-3 in sperm was demonstrated by activation of purified PP1I, and quantitation revealed that immotile caput sperm contained sixfold higher GSK-3 activity than motile caudal sperm. Immunoblot analysis confirmed the expression of GSK-3 in sperm and revealed the occurrence of both the alpha and beta isoforms. Our findings suggest that the higher PP1 activity measured in immotile sperm, presumably due to higher GSK-3 activity, is responsible for holding motility in check. This conclusion was supported by the observation that the phosphatase inhibitors OA and CA, at micromolar and nanomolar levels, respectively, were able to induce motility in completely immotile bovine caput epididymal sperm and to stimulate the kinetic activity of mature caudal sperm. The intrasperm levels of cAMP, pH, and calcium were unaltered by treatment with these inhibitors. The results suggest a biochemical basis for the development and regulation of sperm motility and a possible physiological role for the PP1/I2/GSK-3 system.
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PMID:Sperm motility development in the epididymis is associated with decreased glycogen synthase kinase-3 and protein phosphatase 1 activity. 883 95

Na+, K+-ATPase contributes to the high potassium concentration in the endolymph and the resulting endocochlear potential, which are both essential for the function of the sensory part of the inner ear. Na+, K+-ATPase is present in the stria vascularis and it has lately been suggested that its activity is hormonally regulated. The intracellular signalling system for hormonal short-term regulation of Na+, K+-ATPase activity by phosphorylation in renal tubular cells has been well described. In this study, the presence of the intracellular components of this phosphorylation system in the stria vascularis from guinea-pig has been investigated with immunoblotting. The concentrations found were related to those in renal medullary tissue or the corpus striatum. Protein kinase C was present with isoforms alpha, delta and zeta in the stria vascularis. Calcium- and calmodulin-dependent protein kinase II and protein phosphatase-1 isoforms alpha and gamma were found in the stria vascularis. Protein phosphatase-2B, on the other hand, could not be detected. I-1, an inhibitor of protein phosphatase activity, was present, whereas the phosphatase inhibitor dopamine- and cAMP-regulated phosphoprotein (DARPP-32), was not present in the stria vascularis. These results demonstrate that several intracellular components of the phosphorylation/dephosphorylation system are present in the stria vascularis, and suggest that hormonal short-term regulation of Na+, K+-ATPase activity is also possible in the stria vascularis.
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PMID:Protein kinase and protein phosphatase presence in the stria vascularis. 904 45

Intrarenally formed dopamine induced natriuresis by inhibiting the activity of renal tubular Na/KATPase. This effect is mediated via a complex signal network, which includes inhibition of PP1 via the adenylyl cyclase-PKA-DARPP32 pathway and activation of PKC via the PLA2-arachidonic acid-20HETE pathway. The renal dopamine availability is a major determinant of the natriuretic effect of dopamine and is to a large extent modulated by the activity of COMT. The possibility that regulation of dopamine storage and release influences renal dopamine effects should be considered.
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PMID:The renal dopamine system. 932 36


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