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)

We have studied the role of myosin II light chain phosphorylation in yeast phagocytosis by J774 cells. J774 cells, which are mouse cells of monocyte/macrophage lineage, ingest opsonized yeast particles, and the rate of internalization is linear for 60 min at 37 degrees C. Immunoprecipitation of myosin II from cells labeled with 32P, using an affinity-purified antibody to myosin II purified from J774 cells, demonstrated phosphorylation of both the myosin heavy chain and the 20-kDa light chain (PMLC) prior to the addition of the opsonized yeast. However, the levels of heavy chain and PMLC phosphorylation did not change during the linear phase of yeast uptake by J774 cells. Other experiments demonstrated that the amount of myosin II associated with the cytoskeleton did not change during phagocytosis, further supporting the observation that PMLC phosphorylation does not increase during phagocytosis. In contrast, F-actin increased by 1.6-fold during the linear phase of phagocytosis. Two additional approaches were used to analyze in greater detail the role of myosin II phosphorylation in phagocytosis. First, antibodies to myosin light chain kinase (MLCK), the enzyme that phosphorylates PMLC, were electroinjected into J774 cells. These antibodies, which inhibit MLCK activity, inhibited chemotaxis as previously described but had no effect on phagocytosis. Second, quantitation of phagocytosis and chemotaxis following treatment with the phosphoprotein phosphatase inhibitor okadaic acid demonstrated that chemotaxis was much more sensitive than phagocytosis to okadaic acid treatment; at 0.3 microM okadaic acid, there is a substantial increase in myosin phosphorylation and chemotaxis is inhibited by 60%, whereas phagocytosis is unaffected. These data indicate that PMLC phosphorylation and, by implication, myosin II are not involved in yeast phagocytosis. They also suggest that PMLC phosphorylation displays a high degree of specificity with respect to mediating energy-dependent cellular processes in macrophages.
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PMID:Myosin light chain phosphorylation does not increase during yeast phagocytosis by macrophages. 834 78

Thick filaments are stable assemblies of myosin that are characteristic of specific muscle types from both vertebrates and invertebrates. In general, their structure and assembly require remarkably precise determination of lengths and diameters, structural differentiation and nonequivalence of myosins, a high degree of inelasticity and rigidity, and dynamic regulation of assembly and disassembly in response to both extracellular and intracellular signals. Directed assembly of myosin in which additional proteins function in key roles, therefore, is more likely to be significant than the simple self assembly of myosin into thick filaments. The nematode Caenorhabditis elegans permits a wide spectrum of biochemical, genetic, molecular and structural approaches to be applied to the experimental testing of this hypothesis. Biochemical analysis of C. elegans thick filaments reveals that paramyosin, a homologue of the myosin rod that is the unique product of a single genetic locus, exists as two populations which differ by post-translational modification. The major paramyosin species interacts with the two genetically specified myosin heavy chain isoforms. The minor paramyosin species is organized within the cores of the thick filaments, where it is associated stoichiometrically with three recently identified proteins P20, P28 and P30. These proteins have now been characterized molecularly and contain unique, novel amino acid sequences. Structural analysis of the core shows that seven paramyosin subfilaments are crosslinked by additional internal proteins into a highly rigid tubule. P20, P28 and P30 are proposed to couple the paramyosin subfilaments together into the core tubule during filament assembly. Mutants that affect paramyosin assembly are being characterized for alterations in the core proteins. A fourth protein has been identified recently as the product of the unc-45 gene. Computational analysis of this gene's DNA suggests that the predicted protein may exhibit protein phosphatase and chaperone activities. Genetic analysis shows that three classes of specific unc-45 mutant proteins differentially interact with the two myosins during thick filament assembly. The unc-45 protein is proposed to be a myosin assemblase, a protein catalyst of thick filament assembly.
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PMID:Assemblases and coupling proteins in thick filament assembly. 911 2

Activation of the transcription factor nuclear factor of activated T cells by the calcium-sensitive serine/threonine phosphatase calcineurin has been proposed as one of the molecular mechanisms by which motor nerve activity establishes the slow muscle phenotype. To investigate whether the calcineurin pathway can regulate the large spectrum of slow muscle characteristics in vivo, we treated rats for three weeks with cyclosporin A (an inhibitor of calcineurin). In soleus (slow muscle), but not in plantaris (fast muscle), the proportion of slow myosin heavy chain (MHC-1) and slow sarcoplasmic reticulum ATPase (SERCA2a) was decreased, whereas that of fast MHC (MHC-2A) and fast SERCA1 increased, indicating a slow to fast contractile phenotype transition. Cytosolic isoforms of creatine kinase and lactate dehydrogenase (most abundant in fast fibers), as well as mitochondrial creatine kinase and citrate synthase activities (elevated in fast/oxidative fibers) were dose dependently increased by cyclosporin A treatment in soleus muscle, with no change in plantaris. Calcineurin catalytic subunit was more abundant in soleus muscle fibers compared with plantaris. Taken together these results suggest that the calcineurin pathway co-regulates a set of multigenic protein families involved in the transition between slow oxidative (type I) to fast oxidative (type IIa) phenotype in soleus muscle.
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PMID:Calcineurin Co-regulates contractile and metabolic components of slow muscle phenotype. 1077 82

In this review, the adaptations in myosin heavy chain (MHC) isoform expression induced by chronic reductions in neuromuscular activity (including electrical activation and load bearing) of the intact neuromuscular unit are summarized and evaluated. Several different animal models and human clinical conditions of reduced neuromuscular activity are categorized based on the manner and extent to which they alter the levels of electrical activation and load bearing, resulting in three main categories of reduced activity. These are: 1) reduced activation and load bearing (including spinal cord injury, spinal cord transection, and limb immobilization with the muscle in a shortened position); 2) reduced loading (including spaceflight, hindlimb unloading, bed rest, and unilateral limb unloading); and 3) inactivity (including spinal cord isolation and blockage of motoneuron action potential conduction by tetrodotoxin). All of the models discussed resulted in increased expression of fast MHC isoforms at the protein and/or mRNA levels in slow and fast muscles (with the possible exception of unilateral limb unloading in humans). However, the specific fast MHC isoforms that are induced (usually the MHC-IIx isoform in slow muscle and the MHC-IIb isoform in fast muscle) and the degree and rate of adaptation are dependent upon the animal species and the specific model or condition that is being studied. Recent studies designed to elucidate the mechanisms by which electrical activation and load bearing alter expression of MHC isoforms at the cellular and genetic levels are also reviewed. Two main mechanisms have been proposed, the myogenin:MyoD and calcineurin:NF-AT pathways. Collectively, the data suggest that the regulation of MHC isoform expression involves a complex interaction of multiple control mechanisms including the myogenin:MyoD and calcineurin:NF-AT pathways; however, other intracellular signaling pathways are likely to contribute.
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PMID:Myosin heavy chain isoform expression following reduced neuromuscular activity: potential regulatory mechanisms. 1079 89

Endothelin-1 (ET-1) induces cardiac hypertrophy. Because Ca(2+) is a major second messenger of ET-1, the role of Ca(2+) in ET-1-induced hypertrophic responses in cultured cardiac myocytes of neonatal rats was examined. ET-1 activated the promoter of the beta-type myosin heavy chain gene (beta-MHC) (-354 to +34 base pairs) by about 4-fold. This activation was inhibited by chelation of Ca(2+) and the blocking of protein kinase C activity. Similarly, the beta-MHC promoter was activated by Ca(2+) ionophores and a protein kinase C activator. beta-MHC promoter activation induced by ET-1 was suppressed by pretreatment with the calmodulin inhibitor, W7, the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitor, KN62, and the calcineurin inhibitor, cyclosporin A. beta-MHC promoter activation by ET-1 was also attenuated by overexpression of dominant-negative mutants of CaMKII and calcineurin. ET-1 increased the activity of CaMKII and calcineurin in cardiac myocytes. Pretreatment with KN62 and cyclosporin A strongly suppressed ET-1-induced increases in [(3)H]phenylalanine uptake and in cell size. These results suggest that Ca(2+) plays a critical role in ET-1-induced cardiomyocyte hypertrophy by activating CaMKII- and calcineurin-dependent pathways.
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PMID:Ca2+/calmodulin-dependent kinase II and calcineurin play critical roles in endothelin-1-induced cardiomyocyte hypertrophy. 1080 60

The addition of cyclosporin A (500 ng ml(-1)) - an inhibitor of the Ca2+-calmodulin-regulated serine/threonine phosphatase calcineurin - to primary cultures of rabbit skeletal muscle cells had no influence on the expression of fast myosin heavy chain (MHC) isoforms MHCIIa and MHCIId at the level of protein and mRNA, but reduced the expression of slow MHCI mRNA. In addition, no influence of cyclosporin A on the expression of citrate synthase (CS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was found. The level of enzyme activity of CS was also not affected. When the Ca2+ ionophore A23187 (4 x 10(-7) M) was added to the medium, a partial fast-to-slow transformation occurred. The level of MHCI mRNA increased, and the level of MHCIId mRNA decreased. Cotreatment with cyclosporin A was able to prevent the upregulation of MHCI at the level of mRNA as well as protein, but did not reverse the decrease in MHCIId expression. The expression of MHCIIa was also not influenced by cyclosporin A. Cyclosporin A was not able to prevent the upregulation of CS mRNA under Ca2+ ionophore treatment and failed to reduce the increased enzyme activity of CS. The expression of GAPDH mRNA was reduced under Ca2+ ionophore treatment and was not altered under cotreatment with cyclosporin A. When the myotubes in the primary muscle culture were electrostimulated at 1 Hz for 15 min periods followed by pauses of 30 min, a partial fast-to-slow transformation was induced. Again, cotreatment with cyclosporin A prevented the upregulation of MHCI at the level of mRNA and protein without affecting MHCIId expression. The nuclear translocation of the calcineurin-regulated transcription factor nuclear factor of activated thymocytes (NFATc1) during treatment with Ca2+ ionophore, and the prevention of the translocation in the presence of cyclosporin A, were demonstrated immunocytochemically in the myotubes of the primary culture. The effects of cyclosporin A demonstrate the involvement of calcineurin-dependent signalling pathways in controlling the expression of MHCI, but not of MHCIIa, MHCIId, CS and GAPDH, during Ca2+ ionophore- and electrostimulation-induced fast-to-slow transformations. The data indicate a differential regulation of MHCI, of MHCII and of metabolism. Calcineurin alone is not sufficient to mediate the complete transformation.
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PMID:Calcineurin regulates slow myosin, but not fast myosin or metabolic enzymes, during fast-to-slow transformation in rabbit skeletal muscle cell culture. 1135 Oct 29

Signals that determine fast- and slow-twitch phenotypes of skeletal muscle fibers are thought to stem from depolarization, with concomitant contraction and activation of calcium-dependent pathways. We examined the roles of contraction and activation of calcineurin (CN) in regulation of slow and fast myosin heavy chain (MHC) protein expression during muscle fiber formation in vitro. Myotubes formed from embryonic day 21 rat myoblasts contracted spontaneously, and approximately 10% expressed slow MHC after 12 d in culture, as seen by immunofluorescent staining. Transfection with a constitutively active form of calcineurin (CN*) increased slow MHC by 2.5-fold as determined by Western blot. This effect was attenuated 35% by treatment with tetrodotoxin and 90% by administration of the selective inhibitor of CN, cyclosporin A. Conversely, cyclosporin A alone increased fast MHC by twofold. Cotransfection with VIVIT, a peptide that selectively inhibits calcineurin-induced activation of the nuclear factor of activated T-cells, blocked the effect of CN* on slow MHC by 70% but had no effect on fast MHC. The results suggest that contractile activity-dependent expression of slow MHC is mediated largely through the CN-nuclear factor of activated T-cells pathway, whereas suppression of fast MHC expression may be independent of nuclear factor of activated T-cells.
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PMID:Regulation of myosin heavy chain expression during rat skeletal muscle development in vitro. 1135 38

Mammalian skeletal muscles are a mosaic of different fiber types largely defined by differential myosin heavy chain (MyHC) expression. Little is known about the molecular mechanisms regulating expression of the MyHC gene family members in different fiber types. In this work, we identified several cis- and trans-elements that regulate expression of the three adult fast MyHC genes. Despite multiple DNA-binding motifs for well characterized muscle transcription factors upstream of all three fast MyHC genes, expression of MyoD/Myf-5, calcineurin, or NFAT3 had different effects on the three promoters. MyoD or Myf-5 overexpression preferentially activated the IIb promoter, whereas NFAT or activated calcineurin overexpression preferentially activated the IIa promoter. Calcineurin had a 50-100-fold stimulatory effect on the IIa promoter, and the known downstream effectors of calcineurin (myocyte enhancer factor-2 and NFAT) cannot completely account for this activation. Finally, we identified two elements critical for regulating MyHC-IId/x expression: a 130-base pair enhancer element and a CArG-like element that inhibited IId/x promoter activity in vitro. Thus, we have found specific regulatory pathways that are distinct for the three adult fast MyHC genes. These elements are logical candidates for fiber-specific control of skeletal muscle gene expression in vivo.
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PMID:Different pathways regulate expression of the skeletal myosin heavy chain genes. 1155 68

This study tested the hypothesis that calcineurin signaling is modulated in skeletal muscle cells by fluctuations in nerve-mediated activity. We show that dephosphorylation of NFATc1, MEF2A, and MEF2D transcription factors by calcineurin in all muscle types is dependent on nerve activity and positively correlated with muscle usage under normal weightbearing conditions. With increased nerve-mediated activity, calcineurin dephosphorylation of these targets was found to be potentiated in a way that paralleled the higher muscle activation profiles associated with functional overload or nerve electrical stimulation conditions. We also establish that muscle activity must be sustained above native levels for calcineurin-dependent dephosphorylation of MEF2A and MEF2D to be transduced into an increase in MEF2 transcriptional function, suggesting that calcineurin cooperates with other activity-linked events to signal via these proteins. Finally, examination of individual fiber responses to overload and nerve electrical stimulation revealed that calcineurin-MEF2 signaling occurs in all fiber types but most readily in fibers that are normally least active (i.e. those expressing IIx and IIb myosin heavy chain (MHC)), suggesting that signaling via this phosphatase is also dependent upon the activation history of the muscle cell.
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PMID:Nerve activity-dependent modulation of calcineurin signaling in adult fast and slow skeletal muscle fibers. 1155 50

Nerve activity can induce long-lasting, transcription-dependent changes in skeletal muscle fibers and thus affect muscle growth and fiber-type specificity. Calcineurin signaling has been implicated in the transcriptional regulation of slow muscle fiber genes in culture, but the functional role of calcineurin in vivo has not been unambiguously demonstrated. Here, we report that the up-regulation of slow myosin heavy chain (MyHC) and a MyHC-slow promoter induced by slow motor neurons in regenerating rat soleus muscle is prevented by the calcineurin inhibitors cyclosporin A (CsA), FK506, and the calcineurin inhibitory protein domain from cain/cabin-1. In contrast, calcineurin inhibitors do not block the increase in fiber size induced by nerve activity in regenerating muscle. The activation of MyHC-slow induced by direct electrostimulation of denervated regenerating muscle with a continuous low frequency impulse pattern is blocked by CsA, showing that calcineurin function in muscle fibers and not in motor neurons is responsible for nerve-dependent specification of slow muscle fibers. Calcineurin is also involved in the maintenance of the slow muscle fiber gene program because in the adult soleus muscle, cain causes a switch from MyHC-slow to fast-type MyHC-2X and MyHC-2B gene expression, and the activity of the MyHC-slow promoter is inhibited by CsA and FK506.
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PMID:Calcineurin controls nerve activity-dependent specification of slow skeletal muscle fibers but not muscle growth. 1160 56


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