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:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The substrate specificity of different forms of polycation-stimulated (PCSH, PCSL, and PCSC) phosphorylase phosphatases and of the catalytic subunit of the MgATP-dependent protein phosphatase from rabbit skeletal muscle was investigated. This was done, with phosphorylase a as the reference substrate, using the synthetic phosphopeptides patterned after the phosphorylated sites of pyruvate kinase (type L) (Arg2-Ala-Ser(32P)-Val-Ala (S2), and its Thr(32P) substitute (T4)), inhibitor-1 (Arg4-Pro-Thr(32P)-Pro-Ala (T5), Arg2-Pro-Thr(32P)-Pro-Ala (T1), and its Ser(32P) substitute (S1)), and some modified phosphopeptides (Arg2-Ala-Thr(32P)-Pro-Ala (T2) and Arg2-Pro-Thr(32P)-Val-Ala (T3)), all phosphorylated by cyclic AMP-dependent protein kinase. In addition, casein(Thr-32P), phosphorylated by casein kinase-2, was also tested. The PCS phosphatases show a striking preference for the T4 configuration, PCSC being the least efficient. The catalytic subunit of the MgATP-dependent phosphatase was almost completely inactive toward all these substrates. As shown for the PCSH phosphatase, and comparing with T4, the two proline residues flanking the Thr(P) in T1 and T5, just as in inhibitor-1, drastically imparied the dephosphorylation by lowering the Vmax and not by affecting the apparent Km. The C-terminal proline (as in T2) by itself represents a highly unfavorable factor in the dephosphorylation. The critical effect of the sequence X-Thr(P)-Pro or Pro-Thr(P)-Pro (T1, T2, T5, and inhibitor-1) can be overcome by manganese ions. The additional finding that this is not the case with the Pro-Ser(P)-Pro sequence (S1) suggests that the effect of Mn2+ is highly substrate specific. These observations show the considerable importance of the primary structure of the substrate in determining the specificity of the protein phosphatases.
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PMID:Dephosphorylation of phosphoproteins and synthetic phosphopeptides. Study of the specificity of the polycation-stimulated and MgATP-dependent phosphorylase phosphatases. 302 75

Phosphorylation of protein phosphatase 1 by pp60v-src decreased its activity towards phosphorylase kinase and glycogen synthase as well as towards phosphorylase a. Kinetic experiments indicated that the primary effect of phosphorylation was to increase the Km for each of the substrate proteins. There was little or no change in the Vmax for the reactions. The possibility that phosphorylation of protein phosphatase 1 altered its regulation by inhibitors-1 and -2 was also examined. Phosphorylation of protein phosphatase 1 did not prevent the reversible inhibition of the enzyme by inhibitor-1 or inhibitor-2 nor did it prevent the association of inhibitor-2 with protein phosphatase 1 to form the MgATP-dependent protein phosphatase. Protein phosphatase 1 is not a substrate for pp60v-src when it is complexed with inhibitor-2 to form the inactive MgATP-dependent protein phosphatase. Here we have shown that protein phosphatase 1 is also not phosphorylated by pp60v-src following activation of the MgATP-dependent protein phosphatase with glycogen synthase kinase-3 and MgATP. This indicates that the inability of pp60v-src to phosphorylate protein phosphatase 1 is not due to the change in protein phosphatase 1 conformation which accompanies the inactivation of the MgATP-dependent protein phosphatase. Rather, it appears to be the result of steric hindrance by inhibitor-2. This suggests that the pp60v-src phosphorylation site is closely associated with the inhibitor-2 binding site involved in the formation of the MgATP dependent protein phosphatase. The pp60v-src phosphorylation site was previously localized to a small (Mr less than or equal to 4000) domain which can be selectively degraded by chymotrypsin. Here we have shown that chymotryptic digestion increased the Km of unphosphorylated protein phosphatase 1 for each of the three phosphoprotein substrates used in this study. This effect was similar to that observed after phosphorylation of protein phosphatase 1. These results indicate that the pp60v-src phosphorylation site is in a region of protein phosphatase 1 which influences substrate binding and which may be near the active site.
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PMID:Effects of phosphorylation of protein phosphatase 1 by pp60v-src on the interaction of the enzyme with substrates and inhibitor proteins. 303 Apr 48

Protein phosphatases-1, 2A and 2B have been identified in membrane and soluble fractions of Drosophila melanogaster heads. Similarities between Drosophila and mammalian protein phosphatase-1 included specificity for the beta subunit of phosphorylase kinase, sensitivity to inhibitor-1 and inhibitor-2, inhibition by protamine, retention by heparin-Sepharose and selective interaction with membranes. In addition, an inactive form of protein phosphatase-1, termed protein phosphatase-1I, was detected in the soluble fraction that could be activated by preincubation with MgATP and mammalian glycogen synthase kinase-3. Inhibitor-2 partially purified from Drosophila had an identical molecular mass to its mammalian counterpart, and recombined with mammalian protein phosphatase-1 to form a hybrid protein phosphatase-1I. Similarities between Drosophila and mammalian protein phosphatase-2A included preferential dephosphorylation of the alpha subunit of phosphorylase kinase, insensitivity to inhibitors-1 and -2, activation by protamine, exclusion from heparin-Sepharose and apparent molecular mass. A Ca2+-dependent calmodulin-stimulated protein phosphatase (protein phosphatase-2B) that was inhibited by trifluoperazine was identified in the soluble fraction. The remarkable similarities between Drosophila protein phosphatases and their mammalian counterparts are indicative of strict phylogenetic conservation and demonstrate that the procedures used to classify mammalian protein phosphatases have a wider application. Characterisation of the Drosophila phosphatases will facilitate genetic analysis of dephosphorylation systems and their possible roles in neuronal and behavioural plasticity in Drosophila.
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PMID:The protein phosphatases of Drosophila melanogaster and their inhibitors. 303 Jul 53

Calcineurin, a Ca2+ and calmodulin (CM)-dependent phosphatase, has been shown to be present in high concentrations in the striatum. Using inhibitor-1(phosphorylated by cAMP-dependent protein kinase) as a substrate, we found Ca2+/CM-dependent phosphatase (calcineurin) to be more than 2-fold higher than non-Ca2+/CM-dependent phosphatase in the rat striatum. In order to determine the cellular localization of calcineurin, striatal kainic acid injections were used to destroy neurons whose cell bodies are present at the site of injection. Glutamic acid decarboxylase (GAD) activity was measured as an indicator of destruction of striatal GABA-ergic neurons. After intrastriatal injection of 1 and 2 ug of kainic acid, there was a significant decrease of both calcineurin and GAD. However, injection of 0.5 ug kainic acid into the striatum reduced GAD activity by 81%, but had no effect on calcineurin phosphatase activity. Thus calcineurin does not appear to be equally distributed among all types of striatal neurons, but rather may be concentrated in neurons that are less sensitive to kainic acid than the GABA-ergic neuron.
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PMID:Ca2+/calmodulin-dependent phosphoprotein phosphatase activity of calcineurin in rat striatum: effect of kainic acid lesions. 303 26

Protein phosphatases present in the particulate and soluble fractions of oocytes of the starfish Asterias rubens and Marthasterias glacialis have been classified according to the criteria used for these enzymes from mammalian cells. The major protein phosphatase activity in the particulate fraction had very similar properties to protein phosphatase-1 from mammalian tissues, including preferential dephosphorylation of the beta subunit of phosphorylase kinase, sensitivity to inhibitor-1 and inhibitor-2, inhibition of phosphorylase phosphatase activity by protamine and heparin, and retention by heparin-Sepharose. The major protein phosphatase in the soluble fraction had very similar properties to mammalian protein phosphatase-2A, including preferential dephosphorylation of the alpha subunit of phosphorylase kinase, insensitivity to inhibitors-1 and 2, activation by protamine and heparin, and exclusion from heparin-Sepharose. An acid-stable and heat-stable protein was detected in the soluble fraction of starfish oocytes, whose properties were indistinguishable from those of inhibitor-2 from mammalian tissues. It inhibited protein phosphatase-1 specifically, and its apparent molecular mass on SDS polyacrylamide gels was 31 kDa. Furthermore, an inactive hybrid formed between the starfish oocyte inhibitor and the catalytic subunit of mammalian protein phosphatase-1 could be reactivated by preincubation with MgATP and mammalian glycogen synthase kinase-3. The remarkable similarities between starfish oocyte protein phosphatases and their mammalian counterparts are indicative of strict phylogenetic conservation of these enzymes. The results will facilitate further analysis of the role of protein phosphorylation in the control of starfish oocyte maturation by the hormone 1-methyladenine.
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PMID:Identification of protein phosphatases-1 and 2A and inhibitor-2 in oocytes of the starfish Asterias rubens and Marthasterias glacialis. 304 Mar 98

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein of Mr = 32,000) and phosphatase inhibitor-1, two previously characterized inhibitors of protein phosphatase-1, were identified in both the neostriatum and the substantia nigra. Phosphatase inhibitor-1 was partially purified from bovine caudate nucleus and found to be distinct from DARPP-32 in some of its biochemical properties. The neuronal localization of DARPP-32 and phosphatase inhibitor-1 within the rat neostriatum and substantia nigra was investigated by studying the effects of kainic acid. Injection into the neostriatum of kainic acid, which destroys striatonigral neurons and striatonigral fibers, decreased the amounts of DARPP-32 and phosphatase inhibitor-1 to the same extent, both in the lesioned neostriatum and in the ipsilateral substantia nigra. The specific activity of protein phosphatase-1 in the neostriatum was unaffected by kainic acid. The results indicate that, in rat brain, DARPP-32 and phosphatase inhibitor-1 are both present in striatal neurons and in striatonigral fibers, and that they probably coexist in at least a subpopulation of striatonigral neurons. In contrast, protein phosphatase-1 does not appear to be enriched in any specific neuronal subpopulation in the neostriatum.
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PMID:DARPP-32 and phosphatase inhibitor-1, two structurally related inhibitors of protein phosphatase-1, are both present in striatonigral neurons. 333 43

The complete amino acid sequence of bovine brain DARPP-32, a dopamine- and cyclic AMP-regulated neuronal phosphoprotein, which is a potent and specific inhibitor of the catalytic subunit of protein phosphatase-1, has been determined. The S-14C-carboxymethylated protein was subjected to enzymatic cleavage by endoproteinase Lys-C, endoproteinase Arg-C, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease, and to chemical cleavage by cyanogen bromide. The overlapping sets of peptides were purified by high performance liquid chromatography and subjected to amino acid sequencing by automated Edman degradation to deduce the complete sequence. The protein consists of a single NH2-terminal blocked polypeptide chain of 202 residues, with a calculated molecular mass of 22,591 daltons, excluding the unidentified NH2-terminal blocking group. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis or hydrodynamic measurements. The threonine residue that is phosphorylated by cyclic AMP-dependent protein kinase (Hemmings, H. C., Jr., Williams, K. R., Konigsberg, W. H., and Greengard, P. (1984) J. Biol. Chem. 259, 14486-14490), and that must be phosphorylated for the expression of inhibitory activity, is located at position 34. The molecule contains only 1 cysteine residue and 1 tryptophan residue, at positions 72 and 161, respectively. DARPP-32 is very hydrophilic, and contains a stretch of 16 consecutive acidic residues from position 119 to 134. The predicted secondary structure suggests the presence of 47% alpha-helix, 7% beta-sheet, and 46% random coil, with 11 beta-turns. Comparison of the complete amino acid sequence of bovine DARPP-32 with that of rabbit skeletal muscle protein phosphatase inhibitor-1 revealed a significant amount of sequence identity in the NH2-terminal regions of these two proteins. The active region of inhibitor-1 has been localized to an NH2-terminal fragment (Aitken, A., and Cohen, P. (1982) FEBS Lett. 147, 54-58), the part of the molecule that is most similar to DARPP-32. These data suggest that these two protein phosphatase inhibitors may share a common structural basis for their inhibitory activity and may be related by a common ancestral gene.
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PMID:DARPP-32, a dopamine- and cyclic AMP-regulated neuronal phosphoprotein. Primary structure and homology with protein phosphatase inhibitor-1. 351 Oct 54

The complete primary structure of inhibitor-2, a specific inhibitor of protein phosphatase-1, has been determined. The protein consists of a single polypeptide chain of 203 residues, and has a relative molecular mass of 22835 Da. This molecular mass is significantly lower than earlier estimates based on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The threonyl residue phosphorylated by glycogen synthase kinase-3 is located at position 72. The molecule is very hydrophilic, lacks cysteine residues and the single tryptophanyl and phenylalanyl residues are at positions 46 and 139, respectively. The N-terminal alanyl residue is N-acetylated. Digestion with Staphylococcus aureus V8 proteinase, trypsin, or cleavage with cyanogen bromide, destroyed the biological activity of inhibitor-2, demonstrating that many large fragments (e.g. 1-49, 49-92, 67-101, 108-134, 142-182 and 163-197) are inactive. Digestion with clostripain generated a peptide comprising residues 25-114 which retained 2% of the inhibitory potency of the parent molecule. There is no sequence homology between inhibitor-2 and inhibitor-1.
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PMID:The protein phosphatases involved in cellular regulation. Primary structure of inhibitor-2 from rabbit skeletal muscle. 351 70

The neurotransmitter dopamine has been demonstrated by biochemical, histochemical and immunocytochemical techniques to be unevenly distributed in the mammalian central nervous system. DARPP-32 (dopamine- and cyclic-AMP-regulated phosphoprotein of molecular weight 32,000) is a neuronal phosphoprotein that displays a regional distribution in the mammalian brain very similar to that of dopamine-containing nerve terminals, being highly concentrated in the basal ganglia. The state of phosphorylation of DARPP-32 can be regulated by dopamine and by cyclic AMP in intact nerve cells, suggesting a role for this phosphoprotein in mediating certain of the effects of dopamine on dopaminoceptive cells. The observation that many of the physical and chemical properties of purified DARPP-32 resemble those of phosphatase inhibitor-1 (inhibitor-1), a widely distributed inhibitor of protein phosphatase-1, suggests that DARPP-32 might also function as a phosphatase inhibitor. We report here that DARPP-32 inhibits protein phosphatase-1 at nanomolar concentrations. Moreover, like inhibitor-1, DARPP-32 is effective as an inhibitor in its phosphorylated but not its dephosphorylated form. Thus, the basal ganglia of mammalian brain contain a region-specific neuronal phosphoprotein that is a protein phosphatase inhibitor.
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PMID:DARPP-32, a dopamine-regulated neuronal phosphoprotein, is a potent inhibitor of protein phosphatase-1. 608 60

In liver and muscle the major active phosphorylase and synthase phosphatase activity is associated with the glycogen particle. When we examined the effect of the inhibitor-1 and modulator protein on the enzyme present in crude glycogen fractions from dog liver, the phosphorylase phosphatase was not or only slightly affected. Since the enzyme isolated from the glycogen complex by DEAE-cellulose chromatography could be inhibited by inhibitor-1 as well as the modulator protein, it was assumed that an unknown mechanism or factor present in the glycogen fraction was responsible for this reduced sensitivity of the protein phosphatase. This led to the discovery (7) of the deinhibitor protein which has now been extensively purified from dog liver. The deinhibitor protein was shown to be thermostable, ethanol- and trichloroacetic acid-resistant, but non-dialyzable and it was destroyed by pronase or trypsin. The apparent molecular weight was estimated at about 17,500 in gel filtration, 8,300 in sodium dodecyl sulfate polyacrylamide gel electrophoresis and 5,500 in sucrose density gradient centrifugation, behavior which is consistent with the assumption that the deinhibitor protein may have little ordered structure. Glycogen synthesis requires both phosphorylase and glycogen synthase as dephosphorylated enzymes. The interaction of the deinhibitor protein with the protein phosphatase brings about several effects which, when considered together, could all facilitate the dephosphorylation of glycogen synthase and phosphorylase. The protein phosphatase present in a resuspended glycogen pellet dephosphorylates inhibitor-1 in the absence of Mn2+. This ability of the phosphatase, which is lost during purification of the enzyme, can be restored upon addition of the deinhibitor protein. Owing to the association of the deinhibitor protein with the active phosphatase the enzyme becomes insensitive to inhibition by inhibitor-1 and the modulator protein, and more resistant to the conversion into the FA-ATP,Mg-dependent form, brought about by the modulator protein. During the activation of the ATP,Mg-dependent phosphatase under conditions where kinase FA is rate limiting, the deinhibitor protein increases the level without affecting the rate of activation.
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PMID:Regulation of protein phosphatase activity by the deinhibitor protein. 608 15


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