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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 flip-flop model is a mechanistic model proposed to describe how calmodulin activates enzymes. One prediction based upon this model is that calmodulin-activated enzymes would contain a calmodulin-like binding site which, among other attributes, would bind the peptide melittin. Five purified calmodulin-activated enzymes, namely calcineurin, myosin light chain kinase, phosphorylase b kinase, phosphodiesterase, and NAD kinase, were all found to bind biotinylated melittin and to also bind an antimelittin antibody and biotinylated calmodulins. Using gel blots of crude tissue extracts (rat brain and Arabidopsis), most proteins did not bind any of the probes and thus do not have these characteristics. However, among those which bind any of these probes, a strong correlation was found between those proteins which bind biotinylated calmodulins and those which bind melittin and antimelittin. Gel blots of phosphorylase b kinase demonstrate that the alpha, beta, and gamma subunits all bind calmodulin and melittin. A putative calmodulin-like binding site sequence was identified in eight enzymes or subunits which may play an important role in both melittin binding and calmodulin-dependent regulation of these enzymes.
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PMID:Calmodulin-binding proteins also have a calmodulin-like binding site within their structure. The flip-flop model. 184 67

The distribution of mRNAs encoding two distinct isoforms of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin A), designated calcineurin A alpha and A beta, in the rat brain was examined by using Northern blots and in situ hybridization histochemistry. The mRNAs for calcineurin A alpha and A beta were both unevenly present in the brain and showed different distribution. The differential distribution between calcineurin A alpha and A beta messages suggests that the individual isoforms are involved in specialized neural functions.
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PMID:Differential distribution of the mRNA encoding two isoforms of the catalytic subunit of calcineurin in the rat brain. 184 43

The aim of this work was the identification of the calmodulin-stimulated protein phosphatase, calcineurin, in rat pancreatic islets. For this purpose, a high-affinity calcineurin antibody and the Western blotting technique were used to detect the presence of calcineurin in freshly collagenase-isolated islets. The calcineurin content detected by this method was about 0.30 ng islet (approx. 0.07% of the total islet protein). The subunit composition and Mr of islet calcineurin were similar to those of bovine brain calcineurin. Incubation of nitrocellulose membranes of the Western blotting, containing the islet protein fractions, with 125I-labeled calmodulin and 45Ca2+ demonstrated that the A subunit bound calmodulin, while the B subunit bound Ca2+. The presence of calcineurin in the islets of Langerhans would suggest its possible participation, as a counterpart of the kinases effect, in the regulatory mechanism of insulin secretion.
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PMID:Identification of the calmodulin-regulated protein phosphatase, calcineurin, in rat pancreatic islets. 184 10

In an effort to characterize the second messenger system for LH release, we have previously identified five calmodulin-binding proteins in rat gonadotropes of Mr greater than 205,000, 200,000, 135,000, 60,000, and 52,000. In the present study, we have used a calmodulin overlayer assay combined with Western blotting to determine the molecular identity of three calmodulin-binding proteins in rat gonadotropes: the alpha subunit of spectrin (Mr greater than 205,000), caldesmon (Mr 84,000), and the alpha subunit of calcineurin (Mr 60,000). The Mr greater than 205,000 and Mr 60,000 components or rat pituitary which bind calmodulin are immunoreactive with spectrin and calcineurin antisera, respectively. Rat pituitary also contains an Mr 84,000 component, which is immunoreactive with polyclonal sera and monoclonal antibody raised to chicken gizzard caldesmon (Mr 150,000). Like caldesmon from other sources, the Mr 84,000 component remains soluble after heat treatment and preferentially binds either filamentous actin or calmodulin, depending on the Ca2+ concentration. The three calmodulin-binding proteins were localized specifically in gonadotropes using indirect immunofluorescence microscopy or by Western-blotting cell fractions enriched for gonadotropes. After differential centrifugation of pituitary homogenate, spectrin immunoreactivity was found associated with the nuclear and secretory granule fractions, whereas caldesmon immunoreactivity was seen in the cytosolic fraction and calcineurin in the cytosolic and nuclear fractions. Although the precise role for these proteins remains unknown, the apparent requirement for calmodulin and the small number of calmodulin-binding proteins in the gonadotrope suggest their involvement in mediating GnRH actions.
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PMID:Specific identification and subcellular localization of three calmodulin-binding proteins in the rat gonadotrope: spectrin, caldesmon, and calcineurin. 184 52

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

The 160 and 150 kDa proteins of sarcoplasmic reticulum (SR) are phosphorylated endogenously. The phosphorylation of both proteins has a marked requirement for Ca2+. Half-maximal and maximal phosphorylation was obtained at about 1 nM- and 1 microM-Ca2+ respectively, and a Hill coefficient of about 0.5 was calculated. The phosphorylation is also dependent on NaF as an inhibitor of the SR phosphoprotein phosphatase. The phosphorylation of these proteins is very rapid, and maximal phosphorylation is achieved in less than 15 s. The phosphorylation of the 160 kDa and 150 kDa polypeptides is completely inhibited by 5 mM-MgCl2 and by 75 microM-LaCl3, by very low concentrations of different detergents, and by preincubation of the SR for 2 min at 60 degrees C. The inhibition by Mg2+ is due to stimulation of ATP hydrolysis, thereby decreasing ATP concentration. Different phosphorylated peptides were obtained by digestion with protease V8 of the 160 kDa and 150 kDa protein bands, suggesting that the 160 kDa and 150 kDa proteins are distinct. The two phosphorylated proteins are present in different fractions and preparations of SR, with or without [3H]PN200-110 binding capacity. These and other results suggest that the phosphorylated SR proteins are distinct from the alpha 1 and alpha 2 subunits of the voltage-gated Ca2+ channel of the T-system membranes. Different inhibitors and activators of protein kinase C and calmodulin-dependent protein kinase have no effect on the endogenous phosphorylation of both polypeptides, suggesting that the phosphorylation is regulated solely by Ca2+. A possible regulatory function for this phosphorylation system is described in the accompanying paper [Gechtman. Orr & Shoshan-Barmatz (1991) Biochem. J. 276.97-102].
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PMID:Characterization of Ca(2+)-dependent endogenous phosphorylation of 160,000- and 150,000-Dalton proteins of sarcoplasmic reticulum. 190 35

Calmodulin (CaM) serves as an intracellular Ca2+ receptor in the gonadotrope and appears to mediate GnRH-stimulated gonadotropin release. Recently we have specifically identified three CaM binding proteins of the gonadotrope as calcineurin, caldesmon, and spectrin. Caldesmon (identified by seven polyclonal and a monoclonal antibody, as well as by functional characteristics) appears to be a CaM-regulated, F-actin binding, protein. This 84,000 mol wt component (CaD84) is heat stable and cosediments with F-actin in the absence of Ca2+. In the presence of Ca2+ (greater than 1 microM) this protein disassociates from F-actin and reassociates with calmodulin. We have prepared an antibody which blocks the caldesmon-actin interaction. In the present study, we have loaded this antibody into cells to prevent the (re-)association of caldesmon with F-actin. This treatment synergistically augments the ability of GnRH and other secretogogues (maitotoxin, phorbol myristyl acetate) to stimulate gonadotropin release from the pituitary. This finding, along with the previous observations that GnRH provokes a sufficient rise in intracellular Ca2+ to allow CaM to redistribute and bind proteins which it regulates, suggests a role for caldesmon in GnRH-stimulated gonadotropin release from the pituitary.
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PMID:Caldesmon: a bifunctional (calmodulin and actin) binding protein which regulates stimulated gonadotropin release. 190 32

We have identified a highly active Ca2+ calmodulin-dependent protein kinase in the cytoskeletons of normal (bovine fasciculata) and transformed (Y-1 mouse tumor) adrenal cells. In view of evidence for the involvement of calmodulin and microfilaments in the regulation of cholesterol transport and hence steroidogenesis, it is likely that this kinase is important in this process. The kinase activity was examined for its capacity to phosphorylate endogenous proteins analyzed by one- and two-dimensional gel electrophoresis, in the presence of saturating amounts of Ca2+ (5 mM) and calmodulin (5 microM). Three inhibitors of calmodulin (trifluoperazine, pimozide and W-7) inhibit steroidogenesis and Ca2(+)-calmodulin-dependent phosphorylation kinase activity with similar values for EC50 for the two processes. All three inhibitors inhibit the increased transport of cholesterol to mitochondria in response to ACTH. Two substrates for the kinase (alpha-spectrin and beta-tubulin) were identified and two others (51,000 and 60,000 molecular weight) were tentatively identified as the subunits of the kinase itself in cytoskeletons of both cell types. Calmodulin-binding proteins analyzed by [125I]iodocalmodulin overlay and calmodulin-Sepharose affinity chromatography were also identified in the same cytoskeletons including alpha-spectrin, the Ca2+ calmodulin-dependent phosphatase calcineurin and three that were tentatively identified as the two subunits of the kinase itself and myosin light chain kinase. It is concluded that calmodulin, by binding to the kinase and phosphatase, is capable of influencing the degree of phosphorylation of specific substrates in the cytoskeleton and of forming complexes with spectrin, actin and tubulin. These events may be involved in the regulation of the rate-limiting step of steroidogenesis, i.e. transport of cholesterol to mitochondria.
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PMID:Calcium-calmodulin-dependent phosphorylation of cytoskeletal proteins from adrenal cells. 196 7

Phosphorylation of calmodulin-dependent regulator proteins has been studied in control and CF submandibular glands. Results showed that a 61,000 molecular weight calmodulin-binding protein was less phosphorylated in CF glands than control (p less than 0.001 for difference). The altered calmodulin-binding protein cross-reacted with an antiserum against a known calmodulin-dependent protein phosphatase, calcineurin. An alteration in a protein with calcineurin-like activity in CF epithelial cells would provide a link between defective beta-adrenergic responses and altered Ca2(+)-mediated events in CF and thus might be directly related to the genetic defect.
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PMID:An altered phosphoprotein in cystic fibrosis. 196 59

A 25-amino acid peptide, containing the four protein kinase C (PKC) phosphorylation sites and the calmodulin (CaM) binding domain of the myristoylated alanine-rich C kinase substrate (MARCKS) protein, has been synthesized and used to determine the effects of phosphorylation on its binding and regulation of CaM. PKC phosphorylation of this peptide (3.0 mol of Pi/mol of peptide) produced a 200-fold decrease in its affinity for CaM. PKC phosphorylation of the peptide resulted in its dissociation from CaM over a time course that paralleled the phosphorylation of 1 mol of serine/mol of peptide. The peptide inhibited CaM's binding to myosin light chain kinase and CaM's stimulation of phosphodiesterase and calcineurin. PKC phosphorylation of the peptide resulted in a rapid release of bound CaM, allowing its subsequent binding to myosin light chain kinase (t1/2 = 1.6 min), stimulation of phosphodiesterase (t1/2 = 1.2 min) and calcineurin (t1/2 = 1.7 min). Partially purified MARCKS protein produced a similar inhibition of CaM-phosphodiesterase which was reversed by PKC phosphorylation. PKC phosphorylation of the peptide occurred primarily at serine 8 and serine 12, and phosphorylation of serine 12 regulated peptide affinity for CaM. Thus, PKC phosphorylation of the peptide and the MARCKS protein results in the rapid release of CaM and the subsequent activation of CaM-dependent enzymes. This process might allow for interplay between PKC and CaM-dependent signal transduction pathways.
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PMID:Phosphorylation-dependent binding of a synthetic MARCKS peptide to calmodulin. 200 42


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