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)

Thr-197 phosphate is essential for optimal activity of the catalytic (C) subunit of cAMP-dependent protein kinase enzyme, and, in the C subunit crystal structure, it is buried in a cationic pocket formed by the side chains of His-87, Arg-165, Lys-189, and Thr-195. Because of its apparent role in stabilizing the active conformation of C subunit and its resistance to several phosphatases, the phosphate on Thr-197 has been assumed to be metabolically stable. We now show that this phosphate can be removed from C subunit by a protein phosphatase activity extracted from S49 mouse lymphoma cells or by purified protein phosphatase-2A (PP-2A) with concomitant loss of enzymatic activity. By anion-exchange chromatography, inhibitor sensitivity, and relative activity against glycogen phosphorylase a and C subunit as substrates, the cellular phosphatase resembled a multimeric form of PP-2A. PP-1 was ineffective against native C subunit, but it was able to dephosphorylate Thr-197 in urea-treated C subunit. Accessibility of Thr-197 phosphate to the cellular phosphatase was enhanced by storage of C subunit in a phosphate-free buffer or by inclusion of modest concentrations of urea in the reactions and was reduced by salt concentrations in the physiological range and/or by amino-terminal myristoylation. It is concluded that a multimeric form of PP-2A or a closely related enzyme from cell extracts is capable of removing the Thr-197 phosphate from native C subunit in vitro and could account for significant turnover of this phosphate in intact cells.
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PMID:Dephosphorylation of catalytic subunit of cAMP-dependent protein kinase at Thr-197 by a cellular protein phosphatase and by purified protein phosphatase-2A. 855 May 70

The consequences of site-directed mutagenesis experiments are often anticipated by empirical rules regarding the expected effects of a given amino acid substitution. Here, we examine the effects of "conservative" and "nonconservative" substitutions on the X-ray crystal structures of human recombinant FKBP12 mutants in complex with the immunosuppressant drug FK506 (tacrolimus). R42K and R42I mutant complexes show 110-fold and 180-fold decreased calcineurin (CN) inhibition, respectively, versus the native complex, yet retain full peptidyl prolyl isomerase (PPIase) activity, FK506 binding, and FK506-mediated PPIase inhibition. Interestingly, the structure of the R42I mutant complex is better conserved than that of the R42K mutant complex when compared to the native complex structure, within both the FKBP12 protein and FK506 ligand regions of the complexes, and with respect to temperature factors and RMS coordinate differences. This is due to compensatory interactions mediated by two newly ordered water molecules in the R42I complex structure, molecules that act as surrogates for the missing arginine guanidino nitrogens of R42. The absence of such surrogate solvent interactions in the R42K complex leads to some disorder in the so-called "40s loop" that encompasses the substituent. One rationalization proposed for the observed loss in CN inhibition in these R42 mutant complexes invokes indirect effects leading to a misorientation of FKBP12 and FK506 structural elements that normally interact with calcineurin. Our results with the structure of the R42I complex in particular suggest that the observed loss of CN inhibition might also be explained by the loss of a specific R42-mediated interaction with CN that cannot be mimicked effectively by the solvent molecules that otherwise stabilize the conformation of the 40s loop in that structure.
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PMID:Structure comparison of native and mutant human recombinant FKBP12 complexes with the immunosuppressant drug FK506 (tacrolimus). 856 22

Neurons cultured from neonatal rat hypothalamus and brainstem contain many angiotensin II (Ang II) type 2 (AT2) receptors, and we previously determined that activation of these sites elicited a stimulation of serine/threonine phosphatase 2A (PP2A). Here, we have investigated the effects of Ang II on neuronal mitogen-activated protein (MAP) kinases, potential targets for PP2A. Using in-gel kinase assays and immunoprecipitation analyses we have shown that Ang II (10 nM-1 microM) elicits significant increases in p44(MAPK) (Erk1) and p42(MAPK) (Erk2) activities in cultured neurons, mediated via Ang II type 1 (AT1) receptors. This stimulatory effect of Ang II on Erk1 and Erk2 activities was potentiated by blockade of AT2 receptors with (S)-1-[4-(dimethylamino)-3-methylphenyl]methyl-5-(diphenylacetyl)- 4, 5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acid (PD 123319, 1 microM). Furthermore, the AT2 receptor agonist N-alpha-nicotinoyl-Tyr-Lys-(N-alphaCBZ-Arg)-His-Pro-Ile-OH (CGP42112A) (10-50 nM) caused significant decreases in neuronal Erk1 and Erk2 activities, which were abolished by PD 123319 (1 microM) and by the PP2A inhibitor okadaic acid (3 nM). This indicates that AT1 and AT2 receptors have opposite actions on Erk1 and Erk2 activities in neonatal neurons. Since MAP kinases are involved in the regulation of growth/differentiation and apoptosis, our data may provide an intracellular basis for modulatory effects of Ang II receptors on these processes.
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PMID:Mitogen-activated protein kinases in rat brain neuronal cultures are activated by angiotensin II type 1 receptors and inhibited by angiotensin II type 2 receptors. 866 75

Two short amino acid motifs, WXGDXNXR and PXWCDRXL, define a large family of inositol polyphosphate 5-phosphatases. We tested the importance of seven of these conserved amino acids to substrate binding and catalysis by mutating each to alanine in the platelet 75 kDa inositol polyphosphate 5-phosphatase II (5-phosphatase II). Native and mutant forms of 5-phosphatase II were expressed in baculovirus-infected Sf9 cells, and the recombinant proteins were purified by Mono Q chromatography and studied for enzyme activity. Mutants D476A, N478A, D553A, and R554A had no detectable activity using all four known substrates for this enzyme. Mutants R480A, W551A, and I555A showed greatly reduced hydrolysis of Ins(1,4,5)P3 when compared to native enzyme [Km = 75 microM, Vm = 8300 nmol of Ins(1,4,5)P3 hydrolyzed min-1 (mg of protein)-1]. Mutants W551A and I555A had a Km for Ins(1,4,5)P3 hydrolysis similar to that of the native enzyme (35 microM and 81 microM, respectively), suggesting that these amino acids do not play a role in binding substrate. By contrast, mutant R480A had both increased Km (634 microM) and decreased Vm [855 nmol of Ins(1,4,5)P3 hydrolyzed min-1 (mg of protein)-1]. As judged by measurement of Km, mutant R480A retained normal binding of Ins(1,3,4,5)P4, suggesting that the arginine in motif 2 has a greater role in Ins(1,4,5)P3 binding than in Ins(1,3,4,5)P4 binding. Mutant I555A bound Ins(1,3,4,5)P4 with 8-fold reduced affinity. These mutations markedly reduced 5-phosphatase II hydrolysis of the three other substrates, Ins(1,3,4,5)P4, PtdIns(4,5)P2, and PtdIns(3,4,5)P3. We also tested a mutation comparable to D553A, D460A, in the 110 kDa form of the signaling inositol polyphosphate 5-phosphatase (5SIP110). 5SIP110 D460A had no detectable enzyme activity but retained the ability to bind GRB2. These results are consistent with a role for these conserved amino acids in substrate binding and catalysis.
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PMID:Mutation of the conserved domains of two inositol polyphosphate 5-phosphatases. 867 90

1. The full therapeutic potential of the main immunosuppressive drug, cyclosporin A (CsA), is limited because of its side effects, namely nephrotoxicity and hypertension. Several lines of evidence suggest that the origin of both side effects could be CsA-induced vasoconstriction. However, the underlying molecular mechanisms are not well understood. 2. Diameter measurements of rat isolated mesenteric arteries showed an increase in noradrenaline- and [Arg]8vasopressin-induced vasoconstriction when arteries were pretreated with CsA. 3. Measurements in cultured vascular smooth muscle cells (VSMC) of either cytosolic calcium concentration or of 45Ca2+ efflux showed that CsA potentiated the calcium influx to several vasoconstrictor hormones: [Arg]8vasopressin, angiotensin II, endothelin-1 and 5-hydroxytryptamine. On the other hand, 45Ca2+ efflux in response to thapsigargin, which depletes calcium from intracellular pools, was not potentiated by CsA. 45Ca2+ uptake was not altered by CsA or by any of the analogues tested. 4. Time-course studies in cultured VSMC showed that maximal CsA-induced Ca2+ potentiation occurred after ca. 20 h and this effect was reversed over approximately the next 20 h. 5. To investigate the possible role played by the known intracellular targets of CsA, namely cyclophilin and calcineurin, CsA derivatives with variable potencies with respect to their immunosuppressive activity, were tested on the calcium influx to [Arg]8vasopressin. Derivatives devoid of immunosuppressive activity (cyclosporin H, PSC-833) potentiated calcium signalling, while the potent immunosuppressant, FK520, a close derivative of FK506, and MeVal4CsA, an antagonist of the immunosuppressive effect of CsA did not. The latter compound was unable to reverse the calcium potentiating effect of CsA. 6. Our results show that CsA increases the calcium influx to vasoconstrictor hormones in smooth muscle cells, which presumably increases vasoconstriction. Loading of the intracellular calcium pools appears not to be involved. Experiments with derivatives of CsA and FK520 suggest that interactions with cyclophilins and calcineurin are not the mechanism involved. This indicates, for the first time, that the immunosuppressive activity can be dissociated from the calcium potentiating effect of CsA in vascular smooth muscle.
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PMID:Effect of cyclosporin A and analogues on cytosolic calcium and vasoconstriction: possible lack of relationship to immunosuppressive activity. 879 58

Deletion of the yeast Ser/Thr protein phosphatase PPZ1 results in increased tolerance to sodium and lithium. PPZ1 is also important for cell integrity, as ppz1Delta cells undergo lysis under caffeine stress and PPZ1 overexpression overrides the lytic defect of mutants in the protein kinase C/mitogen-activated protein (MAP) kinase pathway. The PPZ1 protein can be dissected in two halves. The COOH-terminal half is related to type 1 phosphatases, whereas the NH2-terminal half is unrelated to phosphatases and contains a consensus site for N-myristoylation. Several mutated versions of PPZ1 have been constructed and tested for complementation of ppz1Delta mutants. We show that PPZ1 can be myristoylated in vivo and that change of Gly-2 to Ala results in lack of myristoylation and loss of complementation of salt tolerance. Removal of the entire NH2-terminal half results in complete loss of function, although it does not abolish the phosphatase activity of the protein expressed in Escherichia coli. The deletion of a large region of the NH2-terminal half (residues 17-193) does not affect the ability to complement the salt tolerance phenotype but abolish complementation of caffeine sensitivity, whereas the opposite behavior is observed upon removal of residues from 241 to 318. Mutation of Arg-451 to Leu results in both complete loss of function and of phosphatase activity. These results indicates that the NH2-terminal half of the protein contains structural determinants that are specific for certain functions and that the phosphatase activity is required but not sufficient for full PPZ1 function.
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PMID:The NH2-terminal extension of protein phosphatase PPZ1 has an essential functional role. 882 89

An isolated bacterium, identified as a new Sphingomonas species, was demonstrated to contain a novel enzymatic pathway which acted on microcystin LR, the most common cyanobacterial cyclic peptide toxin. Degradation of microcystin LR was mediated by at least three intracellular hydrolytic enzymes. The use of classic protease inhibitors allowed (i) the classification of these enzymes into general protease families and (ii) the in vitro accumulation of otherwise transient microcystin LR degradation products. The initial site of hydrolytic cleavage of the parent cyclic peptide by an enzyme that we designate microcystinase is at the 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-deca-4,6-dienoic acid (Adda)-Arg peptide bond. Two intermediates of microcystin LR enzymatic degradation have been identified; one is linearized (acyclo-) microcystin LR, NH2-Adda-Glu(iso)-methyldehydroalanine-Ala-Leu-beta-methylas partate-Arg-OH, and the other is the tetrapeptide NH2-Adda-Glu(iso)-methyldehydroalanine-Ala-OH. The intermediate degradation products were less active than the parent cyclic peptide; the observed 50% inhibitory concentrations for crude chicken brain protein phosphatase were 0.6 nM for microcystin LR, 95 nM for linear LR, and 12 nM for the tetrapeptide. These linear peptides were nontoxic to mice at doses up to 250 micrograms/kg. Ring opening of the potent hepatotoxin microcystin LR by bacterial microcystinase effectively renders the compound nontoxic by dramatically reducing the interaction with the target protein phosphatase.
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PMID:Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR. 889 99

We have used the patch-clamp technique to study the regulation of the activity of the basolateral small-conductance K+ channel (SK) in the cortical collecting duct (CCD) of the rat kidney. Addition of 50-75 nM calphostin C, an agent which specifically inhibits protein kinase C (PKC), reduced channel activity by 90% in cell-attached patches. In contrast, addition of 1 microM phorbol 12-myristate 13-acetate, a stimulator of PKC, led to addition of "new" K+ channel currents in 9 of 20 patches in the basolateral membrane of the CCD, and the mean increase in NP0, a product of channel number (N) and open probability (Pzero), was 0.90 in these 9 patches. However, application of 1 nM exogenous PKC had no significant effect on channel activity in inside-out patches, suggesting that the PKC effect on the activity of the SK observed in cell-attached patches was not a result of a membrane-delimited action, such as a direct phosphorylation of the SK or closely associated proteins. The effect of calphostin C on the SK can be reversed by addition of either 10 microM S-nitroso-N-acetylpenicillamine, a donor of nitric oxide, or 100 microM 8-bromoguanosine 3',5'-cyclic monophosphate. In addition, the inhibitory effect of calphostin C on the SK was completely abolished by pretreatment of the cells with 1 microM okadaic acid, an inhibitor of protein phosphatase. However, 100 microM N omega-nitro-L-arginine methyl ester, an agent that inhibits nitric oxide synthases (NOS), blocked the SK in cell-attached patches in the presence of okadaic acid, suggesting that the effect of okadaic acid on calphostin C-induced inhibition of the SK was a step before formation of nitric oxide. We conclude that PKC is involved in the stimulation of the SK and that the effect of PKC on the SK may be mediated by regulation of NOS activity in the CCD of the rat kidney.
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PMID:Protein kinase C stimulates the small-conductance K+ channel in the basolateral membrane of the CCD. 894 99

Coiled bodies are conserved subnuclear organelles that contain splicing snRNPs, a subset of nucleolar antigens, and the autoantigen p80 coilin. Most nuclei contain one to five nucleoplasmic coiled bodies, often with one or more located at the nucleolar periphery. Here we show that exposure of mammalian cells to low levels of the specific Ser/Thr protein phosphatase inhibitor, okadaic acid, results in the accumulation of p80 coilin and splicing snRNPs within nucleoli. Mutation of a single serine residue in p80 coilin to aspartate (S202D) also causes coiled bodies and splicing snRNPs to localize within nucleoli when the mutant is transiently transfected and expressed in HeLa cells. Neither okadaic acid nor the S202D coilin mutant causes nucleolar accumulation of serine-arginine-domain proteins. These data indicate that protein dephosphorylation is required to allow normal formation of nucleoplasmic coiled bodies and point to p80 coilin as a substrate whose phosphorylation state may regulate snRNP-nucleolar interactions. The data are consistent with a trafficking mechanism whereby splicing snRNPs cycle through the nucleolus.
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PMID:Inhibition of protein dephosphorylation results in the accumulation of splicing snRNPs and coiled bodies within the nucleolus. 901 10

The function of the GABAA receptor has been studied using the whole cell voltage clamp recording technique in rat cerebellum granule cells in culture. Activation of NMDA-type glutamate receptors causes a reduction in the effect of GABA. Full GABAA receptor activity was recovered after washing out NMDA and NMDA action was prevented in a Mg+2 containing medium. The NMDA effect was also absent when extracellular Ca+2 was replaced by Ba+2 and when 10 mM Bapta was present in the intracellular solution. Charge accumulations via voltage activated Ca+2 channels greater than the ones via NMDA receptors do not cause any reduction in GABAA receptor function, suggesting that Ca+2 influx through NMDA receptor channels is critical for the effect. The NMDA effect was reduced by including adenosine-5'-O-3-thiophosphate (ATP-gamma-S) in the internal solution and there was a reduction in the NMDA effect caused by deltamethrin, a calcineurin inhibitor. Part of the NMDA induced GABAA receptor impairment was prevented by prior treatment with L-arginine. Analogously, part of the NMDA effect was prevented by blockage of NO-synthase activity by N omega-nitro-L-arginine. A combination of NO-synthase and calcineurin inhibitors completely eliminated the NMDA action. An analogous result was obtained by combining the NO-synthase inhibitor with the addition of ATP-gamma-S to the pipette medium. The additivity of the prevention of the NMDA impairment of GABAA receptor by blocking the L-arginine/NO pathway and inhibiting calcineurin activity suggests an independent involvement of these two pathways in the interaction between NMDA and the GABAA receptor. On the one hand Ca+2 influx across NMDA channels activates calcineurin and dephosphorylates the GABAA receptor complex directly or dephosphorylates proteins critical for the function of the receptor. On the other hand, Ca+2 influx activates NO-synthase and induces nitric oxide production, which regulates such receptors via protein kinase G activity.
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PMID:A dual mechanism for impairment of GABAA receptor activity by NMDA receptor activation in rat cerebellum granule cells. 903 53


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