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 cAMP-dependent phosphorylation of sodium channels in rat brain neurons maintained in primary culture. In back phosphorylation studies, cells were treated with drugs to increase intracellular cAMP and sodium channels were solubilized and isolated by immunoprecipitation. Surface and intracellular pools of sodium channels were isolated separately. Purified channels were then phosphorylated with [gamma-32P]ATP by the catalytic subunit of cAMP-dependent protein kinase to incorporate 32P into available cAMP-dependent phosphorylation sites. The amount of 32P incorporated in vitro is inversely proportional to the extent of endogenous phosphorylation. Incubation of cells with forskolin (0.1-100 microM), 8-Br-cAMP (0.1-10 mM), or isobutylmethylxanthine (0.01-1.0 mM) inhibited subsequent incorporation of 32P into isolated sodium channels by 70-80%, indicating that treatment of cells with these drugs had increased endogenous phosphorylation to nearly maximum levels. The phosphopeptides phosphorylated in vivo and in vitro were identical. To examine the magnitude of basal phosphorylation and the extent of stimulated phosphorylation, the amount of 32P incorporated into sodium channels from control and stimulated cells was compared to that from matched samples which had been dephosphorylated with calcineurin. Sodium channels from control cells incorporated approximately 2-fold more 32P after dephosphorylation, indicating that cAMP-dependent sites on the channel are at least 47% phosphorylated in the basal state. Sodium channels from forskolin-treated cells incorporated 7-8-fold more 32P after dephosphorylation, indicating that cAMP-dependent phosphorylation sites are 80-90% phosphorylated after stimulation. Cell surface and intracellular pools of sodium channels were phosphorylated similarly. In cells metabolically labeled with 32P, cell surface sodium channels incorporated 2.7 mol of phosphate/mol of channel. Forskolin stimulated 32P incorporation into sodium channels 1.3-fold, consistent with the results obtained by back phosphorylation. We conclude that the rat brain sodium channel is substantially phosphorylated in both the cell surface and intracellular pools in vivo in unstimulated rat brain neurons, and the extent of phosphorylation is increased to 80-90% of maximum phosphorylation by agents that elevate intracellular cAMP.
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PMID:Cyclic-AMP-dependent phosphorylation of voltage-sensitive sodium channels in primary cultures of rat brain neurons. 244 66

Rat brain sodium channels are phosphorylated at multiple serine residues by cAMP-dependent protein kinase. We have identified soluble rat brain phosphatases that dephosphorylate purified sodium channels. Five separable forms of sodium channel phosphatase activity were observed. Three forms (two, approximately 234 kDa and one, 192 kDa) are identical or related to phosphatase 2A, since they were 85-100% inhibited by 10 nM okadaic acid and contained a 36-kDa polypeptide recognized by a monoclonal antibody directed against the catalytic subunit of phosphatase 2A. Immunoblots performed using antibodies specific for isoforms of the B subunit of phosphatase 2A indicate that the two major peaks of phosphatase 2A-like activity, A1 and B1, are enriched in either B' or B alpha. The remaining two activities (approximately 100 kDa each) probably represent calcineurin. Each was relatively insensitive to okadaic acid, was active only in the presence of CaCl2 and calmodulin, and contained a 19-kDa polypeptide recognized by a monoclonal antibody raised against the B subunit of calcinerurin. Treatment of synaptosomes with okadaic acid to inhibit phosphatase 2A or cyclosporin A to inhibit calcineurin increased apparent phosphorylation of sodium channels at cAMP-dependent phosphorylation sites, as assayed by back phosphorylation. These results indicate that phosphatase 2A and calcineurin dephosphorylate sodium channels in brain, and thus may counteract the effect of cAMP-dependent phosphorylation on sodium channel activity.
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PMID:Identification of soluble protein phosphatases that dephosphorylate voltage-sensitive sodium channels in rat brain. 770 24

We studied the cellular phosphatase inhibitors okadaic acid (OKA), calyculin A, and microcystin on the epithelial sodium channel (ENaC) in A6 renal cells. OKA increased the amiloride-sensitive current after approximately 30 min with maximal stimulation at 1-2 h. Fluctuation analysis of cell-attached patches containing a large number of ENaC yielded power spectra with corner frequencies in untreated cells almost two times as large as in cells pretreated for 30 min with OKA, implying an increase in single channel open probability (P(o)) that doubled after OKA. Single channel analysis showed that, in cells pretreated with OKA, P(o) and mean open time approximately doubled. Two other phosphatase inhibitors, calyculin A and microcystin, had similar effects on P(o) and mean open time. An analog of OKA, okadaone, that does not inhibit phosphatases had no effect. Pretreatment with 10 nM OKA, which blocks protein phosphatase 2A (PP2A) but not PP1 in mammalian cells, had no effect even though both phosphatases are present in A6 cells. Several proteins were differentially phosphorylated after OKA, but ENaC subunit phosphorylation did not increase. We conclude that, in A6 cells, there is an OKA-sensitive phosphatase that suppresses ENaC activity by altering the phosphorylation of a regulatory molecule associated with the channel.
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PMID:Phosphatase inhibitors increase the open probability of ENaC in A6 cells. 1237 79

The Nav1.8 sodium channel isoform, expressed in sensory neurons and implicated in pain responses, is known to be upregulated in Xenopus oocytes by agents that activate protein kinase A. In the absence of exogenous modulators, Nav1.8 channels expressed in oocytes exhibited spontaneous downregulation, so that the amplitudes of peak sodium currents at the end of a 30-min recording period were reduced to 58% of those at the outset of recording with no change in the properties of the expressed channels. Perfusion of oocytes with either cyclosporin A or deltamethrin, considered to be diagnostic inhibitors of the protein phosphatase calcineurin, at 10 microM blocked spontaneous downregulation. These results identify endogenous calcineurin as the mediator of Nav1.8 sodium channel downregulation in oocytes. The use of a calcineurin inhibitor such as cyclosporin A provides an effective means of stabilizing the expression of Nav1.8 sodium channels in oocytes for functional and pharmacological studies.
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PMID:Cyclosporin A and deltamethrin block the downregulation of Nav1.8 sodium channels expressed in Xenopus oocytes. 1533 72

Overexpression of calcineurin in transgenic mouse heart results in massive cardiac hypertrophy followed by sudden death. Sudden deaths are caused by abrupt transitions from sinus rhythm to heart block (asystole) in calcineurin-overexpressing (CN) mice. Preliminary studies showed decreased maximum change in potential over time (dV/dt(max)) of phase 0 of the action potential. Accordingly, the hypothesis was tested that decreased activity of the sodium channel contributes to heart block. Profound decreases in activity of sodium currents (I(Na)) paralleled the changes in action potential characteristics. Progressive age-dependent decreases were observed such that at 42-50 days of life little sodium channel function existed. However, this was not paralleled by decreased protein expression as assessed by immunocytochemistry or by Western blot. Since calcineurin can interact with the ryanodine receptor, we assessed whether chronic in vitro treatment with BAPTA-AM, thapsigargin, and ryanodine could rescue the decrease of I(Na). All of these treatments rescued I(Na) to levels indistinguishable from wild type. The nonspecific PKC inhibitor bisindolylmaleimide I also rescued the decrease of I(Na). To assess whether decreased sodium channel activity contributes to sudden death in vivo, the response to encainide (20 mg/kg) was assessed: 6 of 10 young CN mice died because of asystole, whereas 0 of 10 wild-type mice died (P < 0.01). Moreover, encainide produced exaggerated prolongation of the QRS width in sinus beats before the heart block. Catecholamine tone appears necessary to support life in older CN mice because propranolol (1 mg/kg) triggered asystolic death in five of six CN mice. We conclude that decrease in sodium channel activity is in the common final pathway to asystole in CN mice.
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PMID:Decrease in density of INa is in the common final pathway to heart block in murine hearts overexpressing calcineurin. 1675 Dec 87

Glycogen synthase kinase-3 (GSK-3) is constitutively active in nonstimulated cells, where the majority of its substrates undergo inactivation/proteolysis by phosphorylation. Extracellular stimuli (e.g., insulin) catalyze inhibitory Ser(9)-phosphorylation of GSK-3beta, turning on signaling and causing other biological consequences otherwise constitutively suppressed by GSK-3beta. Regulated and dysregulated activities of GSK-3beta are pivotal to health, disease, and therapeutics (e.g., insulin resistance, neurodegeneration, tumorigenesis, inflammation); however, the underlying mechanisms of multifunctional GSK-3beta remain elusive. In cultured bovine adrenal chromaffin cells, 1) constitutive and negatively-regulated activities of GSK-3beta up- and down-regulated insulin receptor, insulin receptor substrate-1 (IRS-1), IRS-2, and Akt levels via controlling proteasomal degradation and protein synthesis; 2) nicotinic receptor/protein kinase C-alpha (PKC-alpha)/extracellular signal-regulated kinase (ERK) pathway up-regulated IRS-1 and IRS-2 levels, enhancing insulin-induced the phosphoinositide 3-kinase (PI3K)/Akt/GSK-3beta pathway; 3) inhibition of calcineurin by cyclosporin A or FK506 down-regulated IRS-2 level, attenuating insulin-like growth factor-I (IGF-I)-induced ERK and GSK-3beta pathways; and 4) insulin, IGF-I or therapeutics (e.g., lithium) up-regulated the voltage-dependent Na(v)1.7 sodium channel.
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PMID:Drug development targeting the glycogen synthase kinase-3beta (GSK-3beta)-mediated signal transduction pathway: the role of GSK-3beta in the maintenance of steady-state levels of insulin receptor signaling molecules and Na(v)1.7 sodium channel in adrenal chromaffin cells. 1917 6

Striatal-enriched tyrosine phosphatase (STEP) has been identified as a component of physiological and pathophysiological signaling pathways mediated by N-methyl-d-aspartate (NMDA) receptor/calcineurin/calpain activation. Activation of these pathways produces a subsequent change in STEP isoform expression or activation via dephosphorylation. In this study, we evaluated changes in STEP phosphorylation and proteolysis in dissociated cortical neurons after sublethal and lethal mechanical injury using an in vitro stretch injury device. Sublethal stretch injury produces minimal changes in STEP phosphorylation at early time points, and increased STEP phosphorylation at 24 h that is blocked by the NMDA-receptor antagonist APV, the calcineurin-inhibitor FK506, and the sodium channel blocker tetrodotoxin. Lethal stretch injury produces rapid STEP dephosphorylation via NR2B-containing NMDA receptors, but not calcineurin, and a subsequent biphasic phosphorylation pattern. STEP(61) expression progressively increases after sublethal stretch with no change in calpain-mediated STEP(33) formation, while lethal stretch injury results in STEP(33) formation via a NR2B-containing NMDA receptor pathway within 1 h of injury. Blocking calpain activation in the initial 30 min after stretch injury increases the ratio of active STEP in cells and blocks STEP(33) formation, suggesting that STEP is an early substrate of calpain after mechanical injury. There is a strong correlation between the amount of STEP(33) formed and the degree of cell death observed after lethal stretch injury. In summary, these data demonstrate that previously characterized pathways of STEP regulation via the NMDA receptor are generally conserved in mechanical injury, and suggest that calpain-mediated cleavage of STEP(33) should be further examined as an early marker of neuronal fate after stretch injury.
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PMID:In vitro stretch injury induces time- and severity-dependent alterations of STEP phosphorylation and proteolysis in neurons. 2243 60

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