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 actin-activated ATPase activity of myosin II from Acanthamoeba castellanii is inhibited by phosphorylation of 3 serine residues near the carboxyl end of the heavy chain of the molecule. We have purified a protein phosphatase from Acanthamoeba using myosin II as a substrate. This phosphatase has a molecular weight of 39,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point in urea of 5.2. The enzyme also is active against other phosphoserine protein substrates such as turkey gizzard smooth muscle myosin light chain, but not against a synthetic phosphotyrosine protein substrate. It does not hydrolyze ATP or p-nitrophenol phosphate. No effector has been found to increase substantially the activity of the enzyme as isolated, but it is inhibited by ATP, pyrophosphate, and NaF. This inhibition is reduced in the presence of MnCl2. The Mg2+-dependent actin-activated ATPase of myosin II is activated by dephosphorylation of phosphorylated myosin II by the phosphatase. Its broad substrate specificity, molecular weight, and response to protein phosphatase inhibitors suggest that the Acanthamoeba protein phosphatase is a type 2A phosphatase (Cohen, P. (1982) Nature (Lond.) 206, 613-620).
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PMID:Purification of a protein phosphatase from Acanthamoeba that dephosphorylates and activates myosin II. 631 29

1. Two-dimensional electrophoresis has been used to study the extent of phosphorylation of the P light chain of myosin and troponin I in the rabbit beating heart. 2. A procedure has been developed that eliminates endogenous protein phosphatase activity during homogenization and sample preparation for electrophoresis. 3. Evidence has been obtained for two unphosphorylated forms of the P light chain in myosin from the ventricle of the rabbit, guinea pig and cow. 4. In vivo and in the rabbit perfused beating heart about 25% of the P light-chain fraction is in the phosphorylated form. 5. Intervention with adrenaline produced a slight increase in the extent of phosphorylation that reached a maximum after the peak in inotropic response. A similar increase was obtained with ischaemia in the absence of adrenaline. 6. The changes in phosphorylation of the major forms of troponin I identified by electrophoresis occurred after the peak of response to adrenaline and were compatible with previous results.
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PMID:The effect of adrenaline on the phosphorylation of the P light chain of myosin and troponin I in the perfused rabbit heart. 731 29

1. Exogenous arachidonic acid (AA) inhibits the protein phosphatase that dephosphorylates smooth muscle myosin, thus sensitizing the contractile response to Ca2+; it also inhibits voltage-gated Ca2+ channels in smooth muscle. The purpose of the present study was to determine whether endogenous AA is increased by agonists in a manner consistent with its role as a messenger regulating myosin phosphatase and Ca2+ channels. Both AA and diacylglycerol (DAG) were measured in [3H]AA-labelled intact and permeabilized (with staphylococcal alpha-toxin) rabbit femoral arteries stimulated with the alpha 1-adrenergic agonist phenylephrine (PE) (intact and permeabilized smooth muscles) or by guanosine-5'-O-(3-thiotriphosphate (GTP gamma S; permeabilized smooth muscles in which the [Ca2+] was maintained constant). Arachidonic acid mass was determined with gas chromatography and mass spectrometry (GC-MS). 2. In intact smooth muscle, PE increased both AA and DAG levels significantly, to 210 and 145% of baseline values, respectively. Another Ca2+-sensitizing agent, the thromboxane analogue U46619, caused a similar increase in AA and DAG levels in rabbit pulmonary artery. 3. In permeabilized smooth muscle at constant [Ca2+](pCa 6.5) GTP gamma S-induced AA and DAG release preceded force development and GTP gamma S (50 microM, 10 min) increased AA mass to 61-88 microM. 4. Phorbol-12,13-dibutyrate (PDBu), another Ca2+-sensitizing agent, also increased both AA and DAG levels in permeabilized smooth muscle at pCa 6.5, whereas the inactive analogue, 4 alpha-phorbol, did not have a Ca2+-sensitizing effect, nor did it increase AA and DAG levels. 5. In the virtual absence of Ca2+ (pCa > 8) GTP gamma S also increased AA and DAG levels by 3.5- and 1.6-fold, respectively. The effect of free Ca2+ itself on AA and DAG release was modest in the physiological range (pCa 7.0 to pCa 6.0), but pCa 4.5 caused an approximately 3- to 4-fold increase in AA and DAG levels, compared with the levels at pCa 8. In permeabilized ileum smooth muscle maintained at constant [Ca2+] (pCa 6.0), carbachol also significantly increased AA to 1.75 times its original value within 1 min of its application. 6. Our results are consistent with, although do not prove, the roles of AA and DAG as second and/or co-messenger(s) in smooth muscle, while the increases in AA and DAG levels induced by PDBu raise the possibility that they contribute to some of the cellular effects of phorbol esters.
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PMID:Arachidonic acid and diacylglycerol release associated with inhibition of myosin light chain dephosphorylation in rabbit smooth muscle. 756 27

Calponin is a smooth muscle-specific, thin filament-associated protein which has been implicated in the regulation of contraction via its interaction with actin and inhibition of the cross-bridge cycling rate. Calponin is phosphorylated by protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), primarily at S175, with loss of actin binding and inhibition of the actin-activated myosin MgATPase. We previously isolated calponin phosphatase from chicken gizzard smooth muscle and identified it as a type 2A protein phosphatase [Winder et al. (1992) Biochem. J. 286, 197-203]. The methods used to detect phosphatase activity in that study would additionally have detected type 1 and 2C phosphatases, but not type 2B phosphatase (Ca2+/CaM-dependent phosphatase or calcineurin). We have, therefore, examined the expression of type 2B phosphatase in smooth muscle and its ability to dephosphorylate calponin. Western blotting with polyclonal antibodies to the brain enzyme revealed the expression of type 2B phosphatase in chicken gizzard, and immunofluorescence microscopy confirmed the presence of the phosphatase in isolated smooth muscle cells (rabbit and toad stomach). The purified brain phosphatase dephosphorylated calponin (phosphorylated by PKC or CaM kinase II) in a Ca2+/CaM-dependent manner. Dephosphorylation by calcineurin restored actin-binding and actin-activated myosin MgATPase inhibition which had been reduced by PKC-catalyzed phosphorylation. We conclude that calponin dephosphorylation may be catalyzed not only by type 2A phosphatase but also by type 2B phosphatase, raising the possibility that both phosphorylation and dephosphorylation of calponin could be regulated by Ca2+/CaM.
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PMID:Dephosphorylation of calponin by type 2B protein phosphatase. 761 14

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

We report that the C-terminal domain of skeletal muscle dystrophin expressed as a fusion protein with glutathione S-transferase (designated GST-CT-1) is a substrate for Ca2+/calmodulin-dependent phosphorylation and dephosphorylation. GST-CT-1 and GST-CT-1F (GST-CT-1 truncated by 20-25 residues) were phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). The stoichiometries of phosphorylation by CaM kinase II were 1.65 mol of Pi/mol of GST-CT-1 and 0.39 mol of Pi/mol of GST-CT-1F, respectively, suggesting that the principal site(s) of phosphorylation is (are) located in the C-terminal 20-25 residues that are missing from GST-CT-1F. The GST-CT-1 fusion protein was phosphorylated on both serine and threonine residues, whereas GST-CT-1F was phosphorylated only on serine. CaM kinase II-phosphorylated GST-CT-1 and GST-CT-1F were efficiently dephosphorylated by calcineurin, a Ca2+/calmodulin-dependent protein phosphatase (type 2B protein phosphatase). Importantly, calcineurin was found to be associated with a purified sarcolemmal membrane preparation enriched in dystrophin. Type 2A protein phosphatase isolated from smooth muscle (SMP-I) and its catalytic subunit (SMP-ic) also dephosphorylated GST-CT-1, but were less active toward these substrates than was calcineurin. Type 2C phosphatase (SMP-II) and type 1 protein phosphatases [SMP-III, SMP-IV, and myosin-associated phosphatase (PP1M) of smooth muscle and skeletal muscle protein phosphatase 1c] were ineffective in dephosphorylating the C-terminal region of dystrophin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the recombinant C-terminal domain of dystrophin: phosphorylation by calmodulin-dependent protein kinase II and dephosphorylation by type 2B protein phosphatase. 772 17

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

Calponin has been implicated in the regulation of smooth muscle contraction as a result of its ability to inhibit the actin-activated Mg ATPase of smooth muscle myosin. This inhibitory effect is abolished by phosphorylation of calponin by Ca2+/calmodulin-dependent protein kinase II or protein kinase C, and restored following dephosphorylation by a type 2A protein phosphatase. Confocal immunofluorescent images of isolated smooth muscle cells colabeled with antibodies to calponin and actin or to calponin and tropomyosin indicate that calponin is present on thin filaments throughout the cell cytoplasm. Both calponin phosphorylation and myosin light chain phosphorylation increased in intact smooth muscle tissue strips when they contracted in response to carbachol or the phosphatase inhibitor okadaic acid. These results support the hypothesis that calponin phosphorylation-dephosphorylation plays a role in regulating smooth muscle contraction.
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PMID:Calponin and smooth muscle regulation. 776 87

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


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