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)

1. In voltage-clamped whole cells dialysed with GTP, extracellular application of ACh elicits an inwardly rectifying K+ current which subsequently decreases to a steady-state level well below the maximally induced current (desensitization). The mechanism of desensitization of the acetylcholine (ACh)-activated K+ channel current was studied in rat neonatal atrial cells at the single-channel level using the patch-clamp technique. 2. In cell-attached patches with ACh in the pipette, a similar pattern of K+ channel current desensitization was present. Single-channel analyses revealed that the initial rapid decrease in channel activity was associated with progressive shortening of the mean open time (tau o) and prolongation of the mean closed time (tau c) of the K+ channel. 3. In excised, inside-out patches with ACh in the pipette, GTP activated K+ channels with a tau o of approximately 1.0 ms. Addition of ATP to the cytosolic surface resulted in progressive increases in tau o (from 1 to 5 ms) and channel activity. These changes are similar but opposite in direction to those observed during the early phase of ACh-induced channel desensitization in cell-attached patches. 4. The effect of ATP on the channel kinetics was abolished in Mg(2+)-free solution AMP-PNP (adenylyl-imidodiphosphate, a non-hydrolysable analogue of ATP), ADP, CTP (cytidine triphosphate), ITP (inosine triphosphate) or UTP (uridine triphosphate) did not alter the channel kinetics, suggesting that the ATP effect on channel gating probably occurs via phosphorylation by a membrane-bound kinase. H-8 (an isoquinolinesulphonamide derivative which inhibits protein kinases A and C) failed to prevent the action of ATP on the channel. 5. The increases in tau o and channel activity produced by ATP could be completely reversed by an elevation of cytosolic [Ca2+] to 3 x 10(-5) M or above. 6. The effect of Ca2+ on the ATP-induced changes in channel kinetics was blocked by sodium vanadate, a general phosphatase inhibitor. Okadaic acid, an inhibitor of protein phosphatase 1 and 2A, did not block the Ca2+ effect. Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), trifluoroperazine, and calmidazolium, partially blocked the effect of Ca2+. 7. Alkaline phosphatase (20 units/ml) reversed the ATP-induced increases in tau o and channel activity. These results suggest that the ACh-activated K+ channel can be modulated by phosphorylation and dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Modulation of acetylcholine-activated K+ channel function in rat atrial cells by phosphorylation. 165 50

Phosphorylation of the Na+/H+ exchanger in human platelets is apparently controlled by the balancing activities of protein kinase C (PKC) and protein phosphatase (PP). To explore cellular expressions of these activities, we have examined the impact of modulation of PKC and PP on Na+/H+ exchange activity, its pHi set point and intracellular pH (pHi). These parameters were followed spectrofluorimetrically in BCECF-loaded platelets. Phorbol 12-myristate 13-acetate (PMA) and dihexanoylglycerol (DHG), which stimulate PKC, and okadaic acid, which inhibits PP 1 and 2A, elevate the measured parameters in concert, while staurosporine, which inhibits protein kinases, had opposite effects. The stimulatory and inhibitory effects are similarly very rapid, being discerned within seconds. It is concluded that: (a) phosphorylation of the Na+/H+ exchanger is the common origin of the diverse effects of PMA, DHG, okadaic acid and staurosporine, (b) Na+/H+ exchange properties are tightly regulated by phosphorylation and dephosphorylation, and (c) the exchanger plays a major role in pHi regulation in platelets.
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PMID:Modulation of Na+/H+ exchange and intracellular pH by protein kinase C and protein phosphatase in blood platelets. 165 31

The activating factor of ATP.Mg-dependent protein phosphatase (FA) has been identified in brain microtubules. When using purified MAP-2 (microtubule associated protein 2) and tau proteins as substrates, FA could phosphorylate MAP-2 to 16 moles of phosphates per mole of protein with a Km value of 0.4 microM, and tau proteins to 4 moles of phosphates per mole of proteins with a Km value of about 3 microM. When using microtubules as substrates, FA could enhance many-fold the endogenous phosphorylation of many microtubule-associated proteins including MAP-2, tau proteins, and several low-molecular-weight MAPs. In contrast to other reported MAP kinases, such as cAMP-dependent protein kinase and Ca+2/phospholipid-dependent protein kinase, the FA-catalyzed phosphorylation of tau proteins could cause an electrophoretic mobility shift on sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that a dramatic conformational change of tau proteins was produced by FA. Peptide mapping analysis of the phosphopeptides derived from SV8 protease digestion revealed that FA could phosphorylate MAP-2 and tau proteins on at least four specific sites distinctly different from those phosphorylated by cAMP-dependent and Ca+2/phospholipid-dependent MAP kinases. Quantitative analysis further indicated that approximately 19% of the total endogenous kinase activity in brain microtubules was due to FA. Taken together, the results provide initial evidence that the ATP.Mg-dependent protein phosphatase activating factor (FA) is a potent and unique MAP kinase, and may represent one of the major factors involved in phosphorylation of brain microtubules.
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PMID:Identification and characterization of the ATP.Mg-dependent protein phosphatase activator (FA) as a microtubule protein kinase in the brain. 165 23

Chromatography of turkey gizzard extract on Sephacryl S-300 has been shown to fractionate the various smooth muscle phosphatases. We have previously reported the purification and characterization of three of these enzymes, termed smooth muscle phosphatase (SMP)-I, -II, and -IV. Recently, we have purified SMP-III to near homogeneity. Although all of the smooth muscle phosphatases dephosphorylate the isolated myosin light chains, only SMP-III and -IV are active toward intact myosin and, therefore, are most likely to play a direct role in the muscle contraction-relaxation process. SMP-III has a higher molecular weight (390,000), as determined by gel filtration, than the other smooth muscle phosphatases and migrates as single band with a molecular weight of 40,000 in a sodium dodecyl sulfate-polyacrylamide gel. SMP-III is immunologically distinct from SMP-I and -II. It dephosphorylates heavy meromyosin and the isolated myosin light chains at a rapid rate but has low activity toward phosphorylase alpha. The activity of SMP-III is not affected by Ca2+ but is activated by Mn2+.Mg2+ stimulates the activity toward heavy meromyosin but inhibits the myosin light chain phosphatase activity. Attempts to classify SMP-III according to the scheme proposed by Ingebritsen and Cohen (Ingebritsen T. S., and Cohen, P. (1983) Science 221, 331-338) revealed that it is resistant to the heat stable inhibitor-2, suggesting that it is a Type 2 protein phosphatase. However, SMP-III is inhibited by concentrations of okadaic acid which are characteristic of Type 1 protein phosphatases and it binds to heparin-Sepharose like other Type 1 phosphatases. But most interestingly, SMP-III does not dephosphorylate the alpha- or beta-subunits of phosphorylase kinase, a property not reported for any Ser/Thr protein phosphatase.
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PMID:Turkey gizzard smooth muscle myosin phosphatase-III is a novel protein phosphatase. 165 15

Two site-specific antibodies have been prepared by immunizing rabbits with chemically synthesized peptides derived from the partial cDNA-predicted amino acid sequence of extracellular signal-regulated kinase 1 (ERK1), which has been proposed to encode the microtubule-associated protein 2 (MAP2) kinase (Boulton, T. G., Yancopoulos, G. D., Gregory, J. S., Slauer, C., Moomaw, C., Hsu, J., and Cobb, M. H. (1990) Science 249, 64-67). With immunoprecipitation in the presence of sodium dodecyl sulfate (SDS) and Western blotting, an antibody to the peptide containing triple tyrosine residues (alpha Y91) resembling one of the insulin receptor autophosphorylation sites specifically recognized 42- and 44-kDa proteins. On the other hand, an antibody to the peptide corresponding to the COOH terminus portions (alpha C92) of the ERK1 cDNA gene product recognized the 44-kDa protein much more efficiently than the 42-kDa protein. With immunoprecipitation in the absence of SDS, alpha Y91 could barely recognize these two proteins and alpha C92 recognized the 44-kDa protein but failed to recognize the 42-kDa protein. Kinase assays in myelin basic protein (MBP)-containing gel, after SDS-polyacrylamide gel electrophoresis, revealed that insulin or 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated MBP kinase activity in alpha Y91 immunoprecipitates comigrated at molecular mass 42 and 44 kDa. On the other hand, the stimulated MBP kinase activity in alpha C92 immunoprecipitates comigrated only at molecular mass 44 kDa. Insulin stimulated the MBP kinase activity in gels and phosphorylation of these two proteins by greater than 10-fold with a maximal level at 5 min. Insulin and TPA rapidly stimulate the phosphorylation of the 42- and 44-kDa proteins via de novo threonine and tyrosine phosphorylation. Tryptic phosphopeptide mapping analysis of the 42- and 44-kDa proteins, respectively, revealed a single major phosphopeptide containing phosphothreonine and phosphotyrosine, which was common to both insulin- and TPA-stimulated phosphoproteins. Protein phosphatase 2A treatment of these two phosphoproteins caused a complete loss of kinase activity with selective dephosphorylation of phosphothreonine. These data strongly suggest that these two proteins are highly related to the mitogen-activated protein (MAP) kinase with an apparent molecular mass of 42 kDa (Ray, L. B., and Sturgill, T. W. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3753-3757) and that these two immunologically similar but distinct MBP/MAP2 kinases may represent isozymic forms of MBP/MAP2 kinases. These data also demonstrate that insulin and TPA activate MBP/MAP2 kinase activity by de novo phosphorylation of threonine and tyrosine residues via a very similar pathway.
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PMID:Insulin and 12-O-tetradecanoylphorbol-13-acetate activation of two immunologically distinct myelin basic protein/microtubule-associated protein 2 (MBP/MAP2) kinases via de novo phosphorylation of threonine and tyrosine residues. 166 17

The effects of divalent metals, metal chelators (EDTA, EGTA) and sodium dodecyl sulfate were investigated on the phosphatase activity of isolated bovine brain calcineurin assayed in the absence (called intrinsic) and presence of calmodulin. Intrinsic phosphatase was increased by Mn2+, was unaffected by Mg2+, Ca2+, and Ba2+, and was markedly inhibited by Ni2+, Fe2+, Zn2+ and Cu2+. When assayed in the presence of calmodulin, many divalent metals (Ni2+, Zn2+, Pb2+, Cd2+), besides Mn2+, increased modestly the phosphatase activity at low concentrations (10-100 microM) and inhibited it markedly at high concentrations. Ca2(+)-calmodulin stimulated phosphatase activity was antagonized by Ni2+, Zn2+, Fe2+, Cu2+, Pb2+, at low concentrations (50 microM), and by Ba2+, Cd2+ at slightly higher concentrations (greater than 100 microM); Mn2+ and Co2+ (50 microM to 1 mM) in fact augmented it. EDTA and EGTA in a concentration and time dependent fashion inhibited the intrinsic phosphatase activity, particularly that of trypsinized calcineurin. SDS in low concentrations (0.005%) augmented the phosphatase activity and inhibited it at high concentrations. Mn2+ (+/- calmodulin) and Ca2+ only with calmodulin present increased the phosphatase activity assayed with low concentrations of SDS. The EDTA dependent inhibition of intrinsic phosphatase was almost abolished in assays containing SDS. Prior exposure of calcineurin to Mn2+ led to a high activity conformation state of calcineurin that was 'long-lived' or 'pseudo-irreversible'. Such Mn2(+)-activated state of calcineurin exhibited no discernible change in the affinity towards myelin basic protein or its inhibition by trifluoperazine. At alkaline pH, Mg2+ supported the intrinsic phosphatase activity, although to a lesser degree than Mn2+. The latter cation, compared to Mg2+ and Ni2+, was also a more powerful stimulator of the calcineurin phosphatase assayed with histone (III-S) and myosin light chain as substrates.
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PMID:Divalent cation effects on calcineurin phosphatase: differential involvement of hydrophobic and metal binding domains in the regulation of the enzyme activity. 170 Oct 13

Adenylylcyclase activity in the flagella of gametes of Chlamydomonas reinhardtii was inhibited by prior incubation at or below 30 degrees C in the presence of ATP. This decrease did not occur in the absence of ATP, in the presence of the ATP analog 5'-adenylylimidodiphosphate (App(NH)p), or in the presence of ATP plus the protein kinase inhibitor staurosporine (2 microM). If ATP treatment was performed in the absence of an ATP-regenerating system, activity initially declined and subsequently recovered. Incubation of flagella at 45 degrees C in the absence of ATP or incubation at lower temperatures in the presence of either App(NH)p or staurosporine both increased adenylylcyclase activity (over 10-fold) and blocked subsequent ATP-dependent loss of activity at 30 degrees C. This heat-induced activation was prevented by the presence of ATP plus an ATP-regenerating system. Incubation of flagella with [gamma-32P]ATP followed by gel electrophoresis in sodium dodecyl sulfate indicated the presence of endogenous protein kinase and protein phosphatase activities. These data suggest that the flagellar adenylylcyclase in Chlamydomonas gametes is inhibited by phosphorylation and stimulated by dephosphorylation. This mechanism for regulating adenylylcyclase may underlie the rapid increase in cyclic AMP that is induced by flagellar adhesion during fertilization in Chlamydomonas.
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PMID:ATP-dependent regulation of flagellar adenylylcyclase in gametes of Chlamydomonas reinhardtii. 174 89

Dopamine inhibits Na+,K(+)-ATPase activity in several renal tubule segments and thereby regulates urinary Na+ excretion. We now show that a phosphopeptide of 31 amino acids, corresponding to residues 8-38 of the protein phosphatase inhibitor DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32,000), mimics the inhibitory action of dopamine on Na+,K(+)-ATPase activity in renal tubule cells from the ascending limb of the loop of Henle. The dephosphorylated form of the peptide is ineffective. The results indicate that dopamine acts through a protein phosphorylation pathway to regulate the activity of an ion pump. In addition, the data suggest that inhibition of protein phosphatase 1 by phophorylated DARPP-32 is a component of the mechanism by which dopamine regulates urinary Na+ excretion.
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PMID:Phosphorylated Mr 32,000 dopamine- and cAMP-regulated phosphoprotein inhibits Na+,K(+)-ATPase activity in renal tubule cells. 184 76

Glycogen-bound protein phosphatase G from rat liver was transferred from glycogen to beta-cyclodextrin (cycloheptaamylose) linked to Sepharose 6B. After removal of the catalytic subunit and of contaminating proteins with 2 M NaCl, elution with beta-cyclodextrin yielded a single protein on native polyacrylamide gel electrophoresis and two polypeptides (161 and 54 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Several lines of evidence indicate that the latter polypeptides are subunits of the protein phosphatase G holoenzyme. First, these polypeptides were also present, together with the catalytic subunit, in the extensively purified holoenzyme. Also, polyclonal antibodies against these polypeptides were able to bind the holoenzyme. Further, while bound to cyclodextrin-Sepharose, the polypeptides were able to recombine with separately purified type-1 (AMD) catalytic subunit, but not with type-2A (PCS) catalytic subunit. The characteristics of the reconstituted enzyme resembled those of the nonpurified protein phosphatase G. At low dilutions, the spontaneous phosphorylase phosphatase activity of the reconstituted enzyme was about 10 times lower than that of the catalytic subunit, but it was about 1000-fold more resistant to inhibition by the modulator protein (inhibitor-2). In contrast with the free catalytic subunit, the reconstituted enzyme co-sedimented with glycogen, and it was able to activate purified liver glycogen synthase b. Also, the synthase phosphatase activity was synergistically increased by a cytosolic phosphatase and inhibited by physiological concentrations of phosphorylase alpha and of Ca2+.
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PMID:Purification and characterization of the glycogen-bound protein phosphatase from rat liver. 189 24

Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5' region, such that the longer polypeptide (p70 S6 kinase alpha I; calculated Mr 58,946) consists of 525 amino acids, of which the last 502 residues are identical in sequence to the entire polypeptides encoded by the second cDNA (p70 S6 kinase alpha II; calculated Mr 56,153). Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 kinase alpha I cDNA through the utilization of different translational start sites. Although the two p70 S6 kinase polypeptides differ by only 23 amino acid residues, the slightly longer alpha I polypeptide exhibits anomalously slow mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), migrating at an apparent Mr of 90,000 probably because of the presence of six consecutive Arg residues immediately following the initiator methionine. Transient expression of p70 alpha I and alpha II S6 kinase cDNA in COS cells results in a 2.5- to 4-fold increase in overall S6 kinase activity. Upon immunoblotting, the recombinant p70 polypeptides appear as a closely spaced ladder of four to five bands between 65 and 70 kDa (alpha II) and 85 and 90 kDa (alpha I). Transfection with the alpha II cDNA yields only the smaller set of bands, while transfection with the alpha I cDNA generates both sets of bands. Mutation of Met-24 in the alpha I cDNA to Leu or Thr suppresses synthesis of the alpha II polypeptides. Only the p70 alpha I and alpha II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70- and 90-kDa proteins observed in purified rat liver S6 kinase. Moreover, it is the recombinant p70 polypeptides of slowest mobility that coelute with S6 kinase activity on anion-exchange chromatography. The slower mobility and higher enzymatic activity of these p70 proteins is due to Ser/Thr phosphorylation, inasmuch as treatment with phosphatase 2A inactivates kinase activity and increases the mobility of the bands on SDS-PAGE in an okadaic acid-sensitive manner. Thus, the recombinant p70 S6 kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.
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PMID:Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini. 192 62


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