Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P67775 (alpha isoform)
 797 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We examined the immunohistochemical distribution of the two mammalian isoforms of calcineurin catalyic subunits, A alpha and A beta, in central nervous system (CNS) tissues of cows, rats, and humans. Cryostat sections and paraffin sections of parformaldehyde-fixed tissues were stained with antipeptide antibodies for each isoform. The same localization pattern was observed in both cryostat and paraffin sections. In the latter, the intensity of the staining was dramatically enhanced by microwave irradiation. Calcineurin isoforms were localized in a variety of nerve cells but not in neuroglial cells. Their differential expression as the A alpha isoform in the nucleus and the A beta isoform in the cytoplasm was present in a variety of CNS nerve cells, most distinctively in Purkinje cells of the cerebellum and pyramidal cells of the cerebrum, irrespective of species. These results suggest that each isform has distinct intracellular sites of action in CNS neurons and that the phenomenon has been conserved during mammalian evolution.
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PMID:Differential subcellular localization of neural isoforms of the catalytic subunit of calmodulin-dependent protein phosphatase (calcineurin) in central nervous system neurons: immunohistochemistry on formalin-fixed paraffin sections employing antigen retrieval by microwave irradiation. 854 76

The protein phosphatase calcineurin is known to be an essential intracellular signal transducer involved in the TCR-mediated signal transduction pathway and is the common target of the immunosuppressive drugs cyclosporin A (CsA) and FK506. The catalytic subunit of calcineurin exists in multiple isoforms, but their functional differences are not known. It has been assumed that the alpha isoform of calcineurin is the relevant isoform mediating TCR signaling. Recently, calcineurin alpha was knocked out in mice, but no defect in the TCR-mediated IL-2 production was observed, suggesting that another isoform of calcineurin mediates the TCR signal transduction pathway. We have generated specific polyclonal antibodies against the alpha and the beta2 isoforms of calcineurin and examined their distribution in murine tissues and immune cells by immunohistochemical staining and Western blot analysis. We found that the beta2 isoform of calcineurin is predominant in T and B lymphocytes as well as in thymus compared to the alpha isoform, suggesting that the beta2 isoform may play a key role in TCR signaling. Furthermore, we observed that the two isoforms exhibit distinct expression patterns in both kidney and thymus, indicating that the two isoforms of calcineurin have distinct cellular functions. Together, these findings raise the possibility that the nephrotoxicity associated with CsA and FK506 can be reduced by designing novel inhibitors of calcineurin that target specific isoforms of the enzyme.
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PMID:Distinct tissue and cellular distribution of two major isoforms of calcineurin. 939 69

To investigate isoform-specific roles of Ca2+/calmodulin-dependent phosphatase [calcineurin (CaN)] in ischemia-induced cell death, we raised antibodies specific to CaN A alpha and CaN A beta and localized the CaN isoforms in the hippocampal CA1 region of Mongolian gerbils subjected to a 5-min occlusion of carotid arteries. In the nonischemic gerbil, immunoreactions of both isoforms were highly enriched in CA1 regions, especially in the cytoplasm and apical dendrites of CA1 pyramidal neurons. At 4-7 days after the induced ischemia, immunoreactivities of the CaN A alpha isoform in CA1 pyramidal cells were markedly reduced, whereas they were enhanced in the CA1 radiatum and oriens layers. In contrast, CaN A beta immunoreactivities were reduced in all layers of the ischemic CA1 region, whereas they were enhanced in activated astrocytes, colocalizing with glial fibrillary acidic protein. These findings suggest that up-regulation of CaN A alpha in afferent fibers in CA1 and up-regulation of CaN A beta in reactive astrocytes may be involved in neuronal reorganization after ischemic injury.
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PMID:Isoform-specific redistribution of calcineurin A alpha and A beta in the hippocampal CA1 region of gerbils after transient ischemia. 948 52

Catalytic domains of the metalloenzymes protein phosphatases (PPP) 1, 2A and 2B (PP1, PP2A and PP2B, respectively) are homologous to approximately 45%, with the residues in the enzymatic centers strictly conserved. PP1, PP2A and PP2B are abundant in cells and they dephosphorylate serine and/or threonine residues in a variety of proteins serving as cellular phospho switches. The active enzymes work as invariant catalytic subunits PP1c, PP2Ac and PP2Bc, respectively, complexed with diverse regulatory subunits, dependent on the enzymes' specific location and biological function. The crystal structures of PP1c and PP2B (calcineurin) heterotetramer calcineurinA x calcineurinB x FKBP x FK506 have been determined. A comparison of the catalytic subunits of both enzymes indicates their significant structural homology and virtual identity within the catalytic centers, each including a set of conservative amino acids, two metal ions and a phosphate; thus confirming a hypothesis on their common enzymatic mechanisms. The elongated substrate cleft at the active centre is kinked by approximately 120 degrees at the active center in its middle and thus divided into a pre-phospho-Ser/Thr (ligand N-terminal) and a post-phospho-Ser/Thr (ligand C-terminal) section. In PP1c the N-terminal section is highly acidic while in PP2Bc is not. This feature is likely pertinent but not sufficient to the enzymes' selectivity, which is also controlled by regulatory subunits, diverse in various tissues. The metalloenzymes in general and PPP in particular are hard to deal with using theoretical simulations due to parameterization problems for the metal cations. In fact, there are only a few PP1c simulations reported, with the metal di-cations treated quite crudely. This is a preliminary work, in which we introduce and test against some experimental evidence a concept of pseudomolecules of proper geometry, composed of double metal (2Zn2+ or 2Mn2+) cation, and the OH- nuclephile incorporated into the PP1c catalytic site. Both models are associated with either the phosphate (a free enzyme) or the phosphorylated dodecapeptide RRRRPpTPAMLFR, an active fragment (residues 29-40) of a regulatory subunit DARPP-32 inhibitor (PP1c-inhibitor complex); four models total. We have parameterized both pseudomolecules within the AMBER force field. Subsequently, using molecular dynamic in water, we have found the free PP1c subunits to be less stable than the complexed ones and we have speculated on possible reasons for this feature.
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PMID:Molecular modeling of the catalytic domain of serine/threonine phosphatase-1 with the Zn2+ and Mn2+ di-nuclear ion centers in the active site. 1081 8

T lymphocyte activation signals regulate the expression and transactivation function of retinoid X receptor (RXR) alpha through an interplay of complex signaling cascades that are not yet fully understood. We show that cellular Ser/Thr protein phosphatases (PPs) play an important role in mediating these processes. Inhibitors specific for PP1 and PP2A decreased basal expression of RXR alpha RNA and protein in T lymphocyte leukemia Jurkat cells and prevented activation-induced RXR alpha accumulation in these cells. In addition, these inhibitors attenuated the RXR responsive element (RXRE)-dependent transcriptional activation in transient transfection assays. Inhibitors of calcineurin (CN), by contrast, did not have any effect on the basal RXR alpha expression and even augmented activation-induced RXR alpha expression. Expression of a dominant-active (DA) mutant of CN together with a DA mutant of protein kinase C (PKC)theta;, a novel PKC isoform, significantly increased RXRE-dependent transcription. Expression of catalytically inactive PKC theta; or a dominant-negative mutant of PKC theta; failed to synergize with CN and did not increase RXRE-dependent transcription. Expression of a DA mutant of PKC alpha or treatment with PMA was found to attenuate PKC theta; and CN synergism. We conclude that PP1, PP2A, and CN regulate levels and transcriptional activation function of RXR alpha in T cells. In addition, CN synergizes with PKC theta; to induce RXRE-dependent activation, a cooperative function that is antagonized by the activation of the conventional PKC alpha isoform. Thus, PKC theta; and PKC alpha may function as positive and negative modulators, respectively, of CN-regulated RXRE-dependent transcription during T cell activation.
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PMID:Regulation of retinoid X receptor responsive element-dependent transcription in T lymphocytes by Ser/Thr phosphatases: functional divergence of protein kinase C (PKC)theta; and PKC alpha in mediating calcineurin-induced transactivation. 1209 75

We identified a thyroid hormone [3,5,3'-tri-iodothyronine (T(3))]-responsive gene, ZAKI-4, in cultured human skin fibroblasts. It belongs to a family of genes that encode proteins containing a conserved motif. The motif binds to calcineurin and inhibits its phosphatase activity. In the present study, we have demonstrated three different ZAKI-4 transcripts, alpha, beta1 and beta2, in human brain by 5'- and 3'-RACE (rapid amplification of cDNA ends). The alpha transcript was identical with the one that we originally cloned from human fibroblasts and the other two are novel. The three transcripts are generated by alternative initiation and splicing from a single gene on the short arm of chromosome 6. It is predicted that beta1 and beta2 encode an identical protein product, beta, which differs from alpha in its N-terminus. Since alpha and beta contain an identical C-terminal region harbouring the conserved motif, both isoforms are suggested to inhibit calcineurin activity. Indeed, each isoform associates with calcineurin A and inhibits its activity in a similar manner, suggesting that the difference in N-terminus of each isoform does not affect the inhibitory function on calcineurin. An examination of the expression profile of the three transcripts in 12 human tissues revealed that the alpha transcript is expressed exclusively in the brain, whereas beta transcripts are expressed ubiquitously, most abundantly in brain, heart, skeletal muscle and kidney. It was also demonstrated that human skin fibroblasts express both alpha and beta transcripts, raising the question of which transcript is up-regulated by T(3). It was revealed that T(3) markedly induced the expression of alpha isoform but not of beta. This T(3)-mediated increase in the alpha isoform was associated with a significant decrease in endogenous calcineurin activity. These results suggest that the expression of ZAKI-4 isoforms is subjected to distinct hormonal as well as tissue-specific regulation, constituting a complex signalling network through inhibition of calcineurin.
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PMID:Novel human ZAKI-4 isoforms: hormonal and tissue-specific regulation and function as calcineurin inhibitors. 1210 56

The calcineurin (CaN) alpha and beta catalytic subunit isoforms are coexpressed within almost all cell types. The enzymatic properties of CaN heterodimers comprised of the regulatory B subunit (CnB) with either the alpha or beta catalytic subunit were compared using in vitro phosphatase assays. CaN containing the alpha isoform (CnA alpha) has lower K(m) and higher V(max) values than CaN containing the beta isoform (CnA beta) toward the PO4-RII, PO4-DARPP-32(20-38) peptides, and p-nitrophenylphosphate (pNPP). CaN heterodimers containing the alpha or beta catalytic subunit isoform displayed identical calmodulin dissociation rates. Similar inhibition curves for each CaN heterodimer were obtained with the CaN autoinhibitory peptide (CaP) and cyclophilin A/cyclosporin A (CyPA/CsA) using each peptide substrate at K(m) concentrations, except for a five- to ninefold higher IC50 value measured for CaN containing the beta isoform with p-nitrophenylphosphate as substrate. No difference in stimulation of phosphatase activity toward p-nitrophenylphosphate by FKBP12/FK506 was observed. At low concentrations of FKBP12/FK506, CaN containing the alpha isoform is more sensitive to inhibition than CaN containing the beta isoform using the phosphopeptide substrates. Higher concentrations of FKBP12/FK506 are required for maximal inhibition of beta CaN using PO4-DARPP-32(20-38) as substrate. The functional differences conferred upon CaN by the alpha or beta catalytic subunit isoforms suggest that the alpha:beta and CaN:substrate ratios may determine the levels of CaN phosphatase activity toward specific substrates within tissues and specific cell types. These findings also indicate that the alpha and beta catalytic subunit isoforms give rise to substrate-dependent differences in sensitivity toward FKBP12/FK506.
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PMID:Substrate selectivity and sensitivity to inhibition by FK506 and cyclosporin A of calcineurin heterodimers composed of the alpha or beta catalytic subunit. 1213 94

Calcineurin is an important signaling molecule in mesangial cells in vitro and is involved in some manifestations of diabetic nephropathy in vivo. However, calcineurin acts in a cell-specific and tissue-specific manner in the kidney, and mechanisms of specificity are unknown. Three closely related isoforms of the calcineurin A (CnA) subunit are expressed in a tissue-specific manner. This study was undertaken to determine if specificity of calcineurin action is linked to regulation of CnA isoforms in the diabetic kidney. After induction of diabetes with streptozotocin, expression of all three CnA isoforms rapidly increased, primarily in the thick ascending limb of Henle (TAL). After prolonged diabetes, increase specifically of the alpha isoform was observed in collecting ducts (CD) and in endothelial cells of glomeruli. Aquaporin 2 (AQP2), a putative substrate of calcineurin phosphatase in the kidney, is also involved in diabetic nephropathy. Co-localization of CnA isoforms with AQP2 revealed that CnA-alpha is the predominant isoform that associates with AQP2 in the diabetic kidney. Furthermore, inhibition of calcineurin with cyclosporin A (CsA) alters AQP2 localization and phosphorylation in principal cells of CD. Alterations in subcellular localization of AQP2 were parallel with CnA-alpha. Similarly, CsA treatment results in a further increase in urine output compared with diabetes alone, suggesting a functional consequence of inhibiting calcineurin-mediated regulation of AQP2. In conclusion, all three isoforms of CnA are upregulated in the diabetic kidney. Increased expression of CnA-alpha, in particular, is observed in glomeruli and CD and participates in regulation of AQP2 expression, phosphorylation, and function.
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PMID:Differential expression of calcineurin A isoforms in the diabetic kidney. 1515 53

Rat calcineurin (CaN) A alpha isoform (Ppp3ca) cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemia-reperfusion injury. Total RNA was isolated from the heart of the adult Wistar rat, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EGFP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment (3.97 kb) obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno-Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect (CPE) appeared. The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results verified that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A alpha (Ppp3ca) cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.
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PMID:Construction of rat calcineurin A alpha cDNA recombinant adenovirus vector and its identification. 1671 Sep 96

Cadmium, a ubiquitous environmental contaminant, damages several major organs in humans and other mammals. The molecular mechanisms for damage are not known. At high doses (5 mg/kg cadmium chloride or higher), testicular damage in mice, rats, and other rodents includes interstitial edema, hemorrhage, and changes in the seminiferous tubules affecting spermatogenesis. Necrosis is evident by 48 h. The goal of this study was to fine map and identify the cdm gene, a gene that when mutated prevents cadmium-induced testicular toxicity in mouse strains with a mutation in this gene. A serine-threonine phosphatase, calcineurin (CN), subunit A, alpha isoform (Ppp3ca), was one of the seven candidates in the cdm region that was narrowed from 5.6 to 2.0 Mb on mouse chromosome 3. An inhibitor of CN, the immunosuppressant, FK506, prevented cadmium-induced testicular damage in five pathological categories, including vascular endothelial and seminiferous epithelial endpoints. Inductively coupled plasma-mass spectrometry revealed that FK506 protected without lowering the amount of cadmium in the testes. Ppp3ca(-/-) mice were investigated but were found to exhibit endogenous testicular abnormalities, making them an inappropriate model for determining whether the inactivation of the Ppp3ca gene would afford protection from cadmium-induced testicular toxicity. The protection afforded by FK506, found by the current study, indicated that CN is likely to be important in the mechanism of cadmium toxicity in the testis and possibly other organs.
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PMID:FK506, a calcineurin inhibitor, prevents cadmium-induced testicular toxicity in mice. 1778 81


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