Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.16 (calcineurin)
 17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This study explores the role of the calmodulin- and Ca(2+)-sensitive phosphatase calcineurin A in the control of bone resorption by mature osteoclasts. We first cloned full-length calcineurin Aalpha and Abeta cDNA from a rabbit osteoclast library. Sequence analysis revealed an approximately 95 and 86% homology between the amino acid and the nucleotide sequences, respectively, of the two isoforms. The two rabbit isoforms also showed significant homology with the mouse, rat, and human homologs. In situ RT-PCR showed evidence of high levels of expression of calcineurin Aalpha mRNA in freshly isolated rat osteoclasts. Semiquantitative analysis of staining intensity revealed no significant difference in calcineurin Aalpha expression in cells treated with vehicle vs. those treated with the calcineurin (activity) inhibitors cyclosporin A (8 x 10(-7) M) and FK506 (5 x 10(-9) and 5 x 10(-7) M). We then constructed a fusion protein comprising calcineurin Aalpha and TAT, a 12-amino acid-long arginine-rich sequence of the human immunodeficiency virus protein. Others have previously shown that the fusion of proteins to this sequence results in their receptor-less transduction into cells, including osteoclasts. Similarly, unfolding of the TAT-calcineurin Aalpha fusion protein by shocking with 8 M urea resulted in its rapid influx, within minutes, into as many as 90% of all freshly isolated rat osteoclasts, as was evident on double immunostaining with anti-calcineurin Aalpha and anti-TAT antibodies. Pit assays performed with TAT-calcineurin Aalpha-positive osteoclasts revealed a concentration-dependent (10-200 nM) attenuation of bone resorption in the absence of cell cytotoxicity or changes in cell number. TAT-hemaglutinin did not produce significant effects on bone resorption or cell number. The study suggests the following: 1) the 61-kDa protein phosphatase calcineurin Aalpha can be effectively tranduced into osteoclasts by using the TAT-based approach, and 2) the transduced protein retains its capacity to inhibit osteoclastic bone resorption.
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PMID:Molecular cloning, expression, and function of osteoclastic calcineurin Aalpha. 1241 72

It has been difficult to transduce primary cultures of bone cells with proteins of interest. Here, we report the development and validation of a new technology for transduction of osteoblasts and osteoclasts with peptides and moderately sized proteins. Fusion proteins between TAT, an 11 amino acid Arg-rich sequence derived from the HIV protein, and either hemagglutinin or calcineurin Aalpha were synthesized and purified. Exposure of osteoblasts and osteoclasts in primary culture to either TAT-HA or TAT-calcineurin Aalpha resulted in a rapid (within 10 min) intracellular movement of the fusion protein evident on co-immunostaining. Almost 99% of cells were transduced and the fusion protein was retained in approximately 50% of the cells for up to 5 days. TAT did not abolish the functionality of calcineurin Aalpha; the fusion protein stimulated osteoblast differentiation and inhibited osteoclastic resorption. We expect that our studies will provide a firm basis for the future development of TAT fusion proteins for critical molecules involved in bone cell differentiation and function.
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PMID:Transduction of TAT fusion proteins into osteoclasts and osteoblasts. 1244 31

Two of the most commonly used immunosuppressants, cyclosporine A and tacrolimus (FK506), inhibit the activity of a ubiquitously expressed Ca(2+)/calmodulin-sensitive phosphatase, calcineurin. Because both drugs also cause profound bone loss in humans and in animal models, we explored whether calcineurin played a role in regulating skeletal remodeling. We found that osteoblasts contained mRNA and protein for all isoforms of calcineurin A and B. TAT-assisted transduction of fusion protein TAT-calcineurin Aalpha into osteoblasts resulted in the enhanced expression of the osteoblast differentiation markers Runx-2, alkaline phosphatase, bone sialoprotein, and osteocalcin. This expression was associated with a dramatic enhancement of bone formation in intact calvarial cultures. Calcineurin Aalpha(-/-) mice displayed severe osteoporosis, markedly reduced mineral apposition rates, and attenuated colony formation in 10-day ex vivo stromal cell cultures. The latter was associated with significant reductions in Runx2, bone sialoprotein, and osteocalcin expression, paralleled by similar decreases in response to FK506. Together, the gain- and loss-of-function experiments indicate that calcineurin regulates bone formation through an effect on osteoblast differentiation.
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PMID:Calcineurin regulates bone formation by the osteoblast. 1628 45

The transcription factor NFATc1 plays an essential role in transducing signals from RANKL in osteoclast differentiation. To date, however, the specific transcriptional targets of NFATc1 are unknown. Expression of the beta3 integrin is required for normal osteoclast function. We therefore examined the role of NFATc1 in human beta3 integrin expression in osteoclast differentiation. Analysis of the mouse and human beta3 gene promoters revealed considerable sequence homology across a 1.3 kb region upstream of the transcription start site (TSS), with conserved NFAT binding elements present. The region -1242 to +29 (relative to the TSS) was cloned as a luciferase reporter construct (pB3-1.3) and a deletion construct removing to -997 (pB3-1) made. The deletion of 245 bp 5' removed three conserved NFAT sites including a consensus NFAT:AP-1 site. The pB3-1.3 reporter construct was induced by treatment with RANKL in the range 2.5-40 ng/ml and dose-dependently induced by co-transfection with human NFATc1 in RAW264.7 cells. The pB3-1 deletion construct was minimally induced with RANKL treatment and unresponsive to co-transfected NFATc1. Direct NFAT binding to two of the consensus NFAT sites within this 245 bp 5' region was demonstrated by EMSA and supershift with anti-NFAT antibodies. Mutation of two of the conserved NFAT sites in the -1242 to -997 fragment was required to prevent binding. The double NFAT mutant, in the context of the full-length promoter was unresponsive to RANKL treatment or co-transfected NFATc1. We generated cell-permeable TAT-dominant-negative (dn)NFATc1 fusion proteins to assess the effect of blockade of NFAT signaling. Transduction with dnNFAT inhibited RANKL induction of the human beta3 integrin promoter. Involvement of the NFATc1-calcineurin pathway in regulating the human beta3 integrin promoter was further confirmed using the calcineurin pathway inhibitory peptide 11R-VIVIT. Together these results establish the beta3 gene as a direct target of NFATc1 in RANKL-dependent osteoclast formation.
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PMID:NFATc1 regulation of the human beta3 integrin promoter in osteoclast differentiation. 1651 93

The WD repeat scaffolding protein RACK1 can mediate integration of the insulin-like growth factor I receptor (IGF-IR) and integrin signaling in transformed cells. To address the mechanism of RACK1 function, we searched for regulatory proteins that associate with RACK1 in an IGF-I-dependent manner. The serine threonine phosphatase protein phosphatase 2A (PP2A) was found associated with RACK1 in serum-starved cells, and it dissociated immediately upon stimulation with IGF-I. This dissociation of PP2A from RACK1 and an IGF-I-mediated decrease in cellular PP2A activity did not occur in cells expressing either the serine 1248 or tyrosine 1250/1251 mutants of the IGF-IR that do not interact with RACK1. Recombinant RACK1 could bind to PP2A in vitro and restore phosphatase activity to PP2A from IGF-I-stimulated cells. Ligation of integrins with fibronectin or Matrigel was sufficient to facilitate IGF-I-mediated dissociation of PP2A from RACK1 and also to recruit beta1 integrin as PP2A dissociated. By using TAT-fused N-terminal and C-terminal deletion mutants of RACK1, we determined that both PP2A and beta1 integrin interact in the C terminus of RACK1 within WD repeats 4 to 7. This suggests that integrin ligation displaces PP2A from RACK1. MCF-7 cells overexpressing RACK1 exhibited enhanced motility, which could be reversed by the PP2A inhibitor okadaic acid. Small interfering RNA-mediated suppression of RACK1 also decreased the migratory capacity of DU145 cells. Taken together, our findings indicate that RACK1 enhances IGF-I-mediated cell migration through its ability to exclusively associate with either beta1 integrin or PP2A in a complex at the IGF-IR.
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PMID:Insulin-like growth factor I controls a mutually exclusive association of RACK1 with protein phosphatase 2A and beta1 integrin to promote cell migration. 1670 58

Here, we demonstrate that the Ca(2+)/calmodulin-sensitive phosphatase calcineurin is a necessary downstream mediator for osteoclast differentiation. Using quantitative PCR, we detected the calcineurin isoforms Aalpha, Abeta, Agamma (catalytic), and B1 (regulatory) in osteoclast precursor RAW-C3 cells. We found that, although the expression of these isoforms remained relatively unchanged during osteoclast differentiation, there was a profound increase in the expression of their primary substrate for calcineurin, nuclear factor of activated T cells (NFAT)c1. For gain-of-function studies, we incubated osteoclast precursors for 10 min with a calcineurin fusion protein (TAT-calcineurin Aalpha); this resulted in its receptorless influx into >90% of the precursor cells. A marked increase in the expression of the osteoclast differentiation markers tartrate-resistant acid phosphatase (TRAP) and integrin beta(3) followed. In addition, the expression of NFATc1, as well as the alternative substrate for calcineurin, IkappaBalpha, was significantly enhanced. Likewise, transfection with constitutively active NFAT resulted in an increased expression of both TRAP and integrin beta(3). In parallel loss-of-function studies, transfection with dominant-negative NFAT not only inhibited osteoclast formation but also reversed the induction of NFATc1, TRAP, and integrin beta(3) by TAT-calcineurin Aalpha. The expression of these markers was also inhibited by calcineurin Aalpha U1 small nuclear RNA, which significantly reduced calcineurin Aalpha mRNA and protein expression. Consistent with these observations, we observed a reduction in osteoclastogenesis in calcineurin Aalpha(-/-) cells and in osteoclast precursors treated with the calcineurin inhibitors cyclosporin A and FK506. Together, the gain- and loss-of-function experiments establish that calcineurin Aalpha is necessary for osteoclast formation from its precursor and that this occurs via an NFATc1-dependent mechanism.
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PMID:Evidence that calcineurin is required for the genesis of bone-resorbing osteoclasts. 1696 88

Accumulating evidence suggests that upregulation of cyclooxygenase 2 (COX2) in glomerular podocytes promotes podocyte injury. Because Gq signaling activates calcineurin and calcineurin-dependent mechanisms are known to mediate COX2 expression, this study investigated the role of Gqalpha in promoting COX2 expression in podocytes. A constitutively active Gq alpha subunit tagged with the TAT HIV protein sequence was introduced into an immortalized podocyte cell line by protein transduction. This stimulated inositol trisphosphate production, activated an nuclear factor of activated T cells-responsive reporter construct, and enhanced levels of both COX2 mRNA and protein compared with cells treated with a Gq protein lacking the TAT sequence. Induction of COX2 was associated with increased prostaglandin E(2) production and podocyte death, both of which were attenuated by selective COX2 inhibition. In vivo, levels of COX2 mRNA and protein were significantly enhanced in podocytes from transgenic mice that expressed podocyte-targeted constitutively active Gqalpha compared with nontransgenic littermates. These data suggest that Gq-dependent signaling cascades stimulate calcineurin and, in turn, upregulate COX2 mRNA and protein, increase eicosanoid production, and cause podocyte injury.
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PMID:Gq-dependent signaling upregulates COX2 in glomerular podocytes. 1866 30

Micelles were prepared from polymer-peptide block copolymer amphiphiles containing substrates for protein kinase A, protein phosphatase-1, and matrix metalloproteinases 2 and 9. We examine reversible switching of the morphology of these micelles through a phosphorylation-dephosphorylation cycle and study peptide-sequence directed changes in morphology in response to proteolysis. Furthermore, the exceptional uniformity of these polymer-peptide particles makes them amenable to cryo-TEM reconstruction techniques lending insight into their internal structure.
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PMID:Controlling and switching the morphology of micellar nanoparticles with enzymes. 2146 79

Striatal-enriched protein tyrosine phosphatase (STEP) is highly expressed in striatal projection neurons, the neuronal population most affected in Huntington's disease. Here, we examined STEP expression and phosphorylation, which regulates its activity, in N-terminal exon-1 and full-length mutant huntingtin mouse models. R6/1 mice displayed reduced STEP protein levels in the striatum and cortex, whereas its phosphorylation was increased in the striatum, cortex, and hippocampus. The early increase in striatal STEP phosphorylation levels correlated with a deregulation of the protein kinase A pathway, and decreased calcineurin activity at later stages further contributes to an enhancement of STEP phosphorylation and inactivation. Accordingly, we detected an accumulation of phosphorylated ERK2 and p38, two targets of STEP, in R6/1 mice striatum at advanced stages of the disease. Activation of STEP participates in excitotoxic-induced cell death. Because Huntington's disease mouse models develop resistance to excitotoxicity, we analyzed whether decreased STEP activity was involved in this process. After intrastriatal quinolinic acid (QUIN) injection, we detected higher phosphorylated STEP levels in R6/1 than in wild-type mice, suggesting that STEP inactivation could mediate neuroprotection in R6/1 striatum. In agreement, intrastriatal injection of TAT-STEP increased QUIN-induced cell death. R6/2, Tet/HD94, and Hdh(Q7/Q111) mice striatum also displayed decreased STEP protein and increased phosphorylation levels. In Tet/HD94 mice striatum, mutant huntingtin transgene shutdown reestablished STEP expression. In conclusion, the STEP pathway is severely downregulated in the presence of mutant huntingtin and may participate in compensatory mechanisms activated by striatal neurons that lead to resistance to excitotoxicity.
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PMID:Striatal-enriched protein tyrosine phosphatase expression and activity in Huntington's disease: a STEP in the resistance to excitotoxicity. 2163 37

The Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein functions in latently infected cells as an essential participant in KSHV genome replication and as a driver of dysregulated cell growth. To identify novel LANA protein-cell protein interactions that could contribute to these activities, we performed a proteomic screen in which purified, adenovirus-expressed Flag-LANA protein was incubated with an array displaying 4,192 nonredundant human proteins. Sixty-one interacting cell proteins were consistently detected. LANA interactions with high-mobility group AT-hook 1 (HMGA1), HMGB1, telomeric repeat binding factor 1 (TRF1), xeroderma pigmentosum complementation group A (XPA), pygopus homolog 2 (PYGO2), protein phosphatase 2A (PP2A)B subunit, Tat-interactive protein 60 (TIP60), replication protein A1 (RPA1), and RPA2 proteins were confirmed in coimmunoprecipitation assays. LANA-associated TIP60 retained acetyltransferase activity and, unlike human papillomavirus E6 and HIV-1 TAT proteins, LANA did not reduce TIP60 stability. The LANA-bound PP2A B subunit was associated with the PP2A A subunit but not the catalytic C subunit, suggesting a disruption of PP2A phosphatase activity. This is reminiscent of the role of simian virus 40 (SV40) small t antigen. Chromatin immunoprecipitation (ChIP) assays showed binding of RPA1 and RPA2 to the KSHV terminal repeats. Interestingly, LANA expression ablated RPA1 and RPA2 binding to the cell telomeric repeats. In U2OS cells that rely on the alternative mechanism for telomere maintenance, LANA expression had minimal effect on telomere length. However, LANA expression in telomerase immortalized endothelial cells resulted in telomere shortening. In KSHV-infected cells, telomere shortening may be one more mechanism by which LANA contributes to the development of malignancy.
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PMID:A protein array screen for Kaposi's sarcoma-associated herpesvirus LANA interactors links LANA to TIP60, PP2A activity, and telomere shortening. 2237 92


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