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)

A covalent adduct of norchlorpromazine (CAPP) and calmodulin is a very potent antagonist of calmodulin activation of several enzymes. The phenothiazine-calmodulin complex (CAPP-calmodulin) acts as a pure antagonist with phosphodiesterase and myosin kinase or a partial agonist with the phosphoprotein phosphatase, calcineurin. Because of its potency and the selectivity inherent to its calmodulin moiety, CAPP-calmodulin should be a uniquely useful probe of calmodulin actions.
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PMID:CAPP-calmodulin: a potent competitive inhibitor of calmodulin actions. 631 84

In this study, we examined the role of insulin, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) cascade in activation of protein phosphatase-1 (PP-1) by using three complementary approaches. First, differentiated L6 cells were acutely exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA, 400 nM) to activate PKC. In these cells, TPA caused 32% stimulation of PP-1 activity. The PP-1 stimulation by TPA was comparable to stimulation by insulin (t1/2 = 1 min and EC50 = 5 nM) with a maximum effect in 5 min. The effects of insulin and TPA were not additive. Insulin and TPA also stimulated MAPK (> 2-fold increase over basal, with myelin basic protein as a substrate). ML-9, a myosin light chain kinase inhibitor, blocked the effects of insulin and TPA on both MAPK and PP-1 activation. In the second approach, PKC was down-regulated by chronic treatment with TPA. In these cells subsequent effects of insulin on MAPK and PP-1 activation were blocked, without an effect on basal enzyme levels. In the third approach, two selective inhibitors of PKC, calphostin and chelerythrine chloride, were used to inhibit PKC. These inhibitors completely prevented insulin and TPA stimulation of MAPK and PP-1 and blocked insulin-induced translocation of PKC to the plasma membranes. We conclude that PKC plays an important role in insulin stimulation of PP-1 via the activation of MAPK cascade.
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PMID:Stimulation of protein phosphatase-1 activity by phorbol esters. Evaluation of the regulatory role of protein kinase C in insulin action. 751 82

A calmodulin-like protein, which is identical in size and 85% identical to vertebrate calmodulin, was recently identified by 'subtractive hybridization' comparison of transcripts expressed in normal versus transformed human mammary epithelial cells. Unlike the ubiquitous distribution of calmodulin, calmodulin-like protein expression is restricted to certain epithelial cells, and appears to be modulated during differentiation. In addition, calmodulin-like protein levels are often significantly reduced in malignant tumor cells as compared to corresponding normal epithelial cells. The current studies compare calmodulin-like protein functions with those of calmodulin. We find that calmodulin-like protein activation of multifunctional Ca2+/calmodulin-dependent protein kinase II (calmodulin kinase II) is equivalent to activation by calmodulin, but that four other calmodulin-dependent enzymes, cGMP phosphodiesterase, calcineurin, nitric-oxide synthase, and myosin-light-chain kinase, display much weaker activation by calmodulin-like protein than by calmodulin. In the case of myosin-light-chain kinase, calmodulin-like protein competitively inhibits calmodulin activation of the enzyme with a Ki value of 170 nM. Thus, calmodulin-like protein may have evolved to function as a specific agonist of certain calmodulin-dependent enzymes, and/or as a specific competitive antagonist of other calmodulin-dependent enzymes.
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PMID:Selective activation and inhibition of calmodulin-dependent enzymes by a calmodulin-like protein found in human epithelial cells. 752 42

In the presence of Ca2+ and calmodulin (CM), purified smooth muscle myosin light chain kinase (MLCKase) was found to undergo autophosphorylation at a rate that was about 200-fold slower than its catalytic activity. Up to 1.7 mol of phosphate were incorporated per mole of kinase. Lower levels of incorporation could be correlated with the presence of an endogenous protein phosphatase which could be inhibited with okadaic acid or Microcystin-LR. The major autophosphorylation site was identified as Thr-863 or Thr-865 and was located on the 24-kDa C-terminal fragment of the kinase. In addition, there was a relatively low and variable contribution of a Ca/CM-independent autophosphorylation at Ser-814 or Ser-815. The initial autophosphorylation rates and maximal incorporation levels were highest at a molar ratio of 2 MLCKase to 1 CM and were inhibited at higher CM levels. This indicated that binding of one molecule of the kinase apoenzyme by a CM-kinase complex was necessary for the reaction to occur. Kinetic analysis of the autophosphorylation reaction was consistent with this interpretation and indicated a second-order intermolecular process that included MLCKase dimerization or oligomerization. In contrast, the low Ca/CM-independent contribution was of intramolecular type since it did not depend on the kinase concentration. The autophosphorylation appeared to be involved in a relatively slow modification of the oligomeric properties of the kinase leading to a 2-4-fold amplification of the kinase catalytic activity which followed its activation by CM. Oligomerization and dimerization of the kinase was independently demonstrated by light scattering measurements.
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PMID:Calmodulin-dependent autophosphorylation of smooth muscle myosin light chain kinase: intermolecular reaction mechanism via dimerization of the kinase and potentiation of the catalytic activity following activation. 754 20

Thrombin-induced cultured bovine endothelial cell (EC) gap formation and albumin permeability is initiated by contraction, which is dependent upon myosin light chain kinase-mediated myosin light chain (MLC) phosphorylation. MLC are then rapidly dephosphorylated (J. G. N. Garcia, H. W. Davis, and C. E. Patterson, J. Cell. Physiol. 163: 510-522, 1995), suggesting a role for MLC dephosphorylation in regulation of EC barrier function. Therefore, we studied the effect of semiselective protein phosphatase (PPase) inhibitors, calyculin A and okadaic acid, on MLC phosphorylation status, myosin-associated PPase activity, and EC monolayer permeability. Calyculin A (0.1-10 nM), but not okadaic acid (1-100 nM) produced significant dose-dependent enhancement of both MLC phosphorylation (three- to four-fold) and EC permeability (eightfold). EC homogenates were utilized to assess Ser/Thr PPase activities using either [32P]phosphorylase A or 32P-labeled skeletal MLC as substrates. Calyculin A at 5 nM (sufficient to inhibit type 1 and type 2A PPase) produced approximately 95% inhibition of all EC PPase activity against both substrates, whereas 2 nM okadaic acid (selective for PPase 2A) only partially inhibited EC PPase activity (40-60%). Fractionation of EC homogenates produced a supernatant fraction containing < 10% of total myosin and a pellet fraction with > 90% of total myosin. PPase activity in the myosin-enriched pellet was insensitive to 2 nM okadaic acid (0% inhibition) but sensitive to 5 nM calyculin (> 95% inhibition). Immunoreactive PPase 1 was present in both fractions, whereas PPase 2A was present only in the myosin-depleted fraction. We conclude that a type 1 myosin-associated PPase is involved in regulation of EC contractility and barrier function.
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PMID:Regulation of endothelial cell gap formation and barrier function by myosin-associated phosphatase activities. 763 21

In summary, phosphorylation of the regulatory light chain of myosin by Ca2+/CaM-dependent MLCK plays an important role in smooth muscle contraction. Although there have been major advances in our understanding of the regulation and physiological functions of contractile proteins in smooth muscle in recent years, very little information exists on the functional status of these proteins in human myometrium during pregnancy. The simple view that contractile force in smooth muscle is proportionate to cytoplasmic Ca2+ concentrations (Ca2+i) and myosin light chain phosphorylation is now more complex as more experiments provide insights into mechanisms of regulation of the contractile elements. MLCK can be phosphorylated, which desensitizes its activation by Ca2+/CaM, and protein phosphatase activity toward myosin may also be regulated. Examples in smooth muscle tissue are sparse, and the different mechanisms by which these processes may be adapted in uterine smooth muscle during pregnancy are not well-defined. Much research is needed to define further the cellular, biochemical, and molecular basis for these physiological processes involved in the regulation of uterine smooth muscle contraction and relaxation.
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PMID:Myosin phosphorylation and the control of myometrial contraction/relaxation. 775 9

Smooth muscle contraction is regulated primarily by the reversible phosphorylation of myosin by myosin light chain kinase. Secondary mechanisms that might modulate contractility are phosphorylation-dephosphorylation of myosin light chain kinase and thin-filament proteins, caldesmon and calponin. Purification of several protein phosphatases that are active toward myosin light chains and (or) myosin and heavy meromyosin from smooth muscles has been reported. All the cytosolic turkey gizzard smooth muscle phosphatases, termed SMP-I, -II, -III, and -IV, dephosphorylate myosin light chains rapidly, but only SMP-III and -IV are active toward myosin and heavy meromyosin, suggesting that SMP-III and -IV might be directly involved in the relaxation of smooth muscle. SMP-III and -IV exhibit properties typical of type 1 protein phosphatases following tryptic digestion. These enzymes appear to share structural similarity with myofibrillar phosphatase PP1M. Purified calponin phosphatase and caldesmon phosphatase from chicken gizzards are structurally and immunologically identical with SMP-I, a type 2A protein phosphatase. SMP-I dephosphorylates calponin faster than it does caldesmon, and has much higher activity toward these substrates than SMP-II, -III, and -IV. Thus, one role for SMP-I might be to regulate the activities of caldesmon and calponin. Since SMP-I is active toward myosin light chain kinase, it might also modulate this enzyme.
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PMID:Smooth muscle phosphatases: structure, regulation, and function. 776 89

The sites of action of many chemical agents that modify the contraction of smooth muscle are in the smooth muscle membrane. However, a few agents, such as calmodulin inhibitors and protein kinase inhibitors, interact directly with contractile elements of the actomyosin system so as to modify smooth muscle contraction. Here, we describe experimental procedures that are applicable for the screening of smooth muscle relaxants with this mode of action. Myosin B was extracted from chicken gizzard smooth muscle. Because myosin B was a crude preparation of smooth muscle actomyosin, it consisted of regulatory proteins of calmodulin, myosin light chain kinase and protein phosphatase in addition to the contractile proteins of actin and myosin. Interaction of chemical agents with these proteins could be detected by measuring the Mg-ATPase activity of the myosin B preparation. Then we examined whether the agents that altered the ATPase activity was associated with changes in phosphorylation of myosin light chain. If the levels are altered, the agents may interact with the regulatory protein(s). If not, the site of their action was in the contractile proteins. The analysis with these respective proteins will be also described.
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PMID:[Studies on agonists and antagonists of smooth muscle contraction by the use of an actomyosin preparation]. 782 22

Smooth muscle cells in the walls of many organs are vital for most bodily functions, and their abnormalities contribute to a range of diseases. Although based on a sliding-filament mechanism similar to that of striated muscles, contraction of smooth muscle is regulated by pharmacomechanical as well as by electromechanical coupling mechanisms. Recent studies have revealed previously unrecognized contractile regulatory processes, such as G-protein-coupled inhibition of myosin light-chain phosphatase, regulation of myosin light-chain kinase by other kinases, and the functional effects of smooth muscle myosin isoforms. Abnormalities of these regulatory mechanisms and isoform variations may contribute to diseases of smooth muscle, and the G-protein-coupled inhibition of protein phosphatase is also likely to be important in regulating non-muscle cell functions mediated by cytoplasmic myosin II.
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PMID:Signal transduction and regulation in smooth muscle. 796 67

Q10 values of the protein phosphatases that can dephosphorylate the regulatory light chain of smooth muscle myosin were determined. Six phosphatases were examined, i.e. skeletal muscle protein phosphatase 1c; protein phosphatase 2Ac; smooth muscle phosphatases (SMP) I, II, and IV; and myosin-associated protein phosphatase (MAP phosphatase). Among them, SMP-IV and MAP phosphatase, which can dephosphorylate intact smooth muscle myosin, showed extremely high Q10 values (5.3 and 5.2, respectively). On the other hand, the Q10 values of other tested phosphatases were within the range of the normal enzyme reaction (Q10 = 2.0). The rate of dephosphorylation of the myosin light chain in alpha-toxin-skinned strips was measured at different temperatures. The results provided a Q10 of 5.1, which was quite similar to those values obtained for SMP-IV and MAP phosphatase. These results suggest that the physiological myosin light chain phosphatases are SMP-IV and/or MAP phosphatase, i.e. type 1 protein phosphatases. The temperature dependence of maximum force, the steady-state extent of myosin light chain phosphorylation, and the relaxation rate of alpha-toxin-permeabilized rabbit portal vein smooth muscle strips were measured. Both maximum force and the extent of myosin light chain phosphorylation were significantly higher at lower temperature (15 degrees C) than at higher temperature (25 degrees C) under all pCa conditions tested, i.e. > 8, 6.3, and 5. The temperature dependence of the relaxation rate was much steeper (decreased 4 times by lowering the temperature from 25 to 15 degrees C) than that of the initial rate of increase in force development (decreased 1.4 times by lowering the temperature from 25 to 15 degrees C). These results are consistent with the Q10 values of myosin light chain phosphatases (Q10 = 5) and myosin light chain kinase (Q10 = 1.7) and further show that the smooth muscle type 1 phosphatases are responsible for the dephosphorylation of smooth muscle myosin in situ.
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PMID:Correlation between high temperature dependence of smooth muscle myosin light chain phosphatase activity and muscle relaxation rate. 811 26


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