Cyclosporin-mediated inhibition of bovine calcineurin by cyclophilins A and B.

Swanson, Selene K.; Born, Timothy L.; Zydowsky, Lynne D.; Cho, Hyeongjin; Chang, Howard Y.; Walsh, Christopher T.; Rusnak, Frank

doi:10.1073/pnas.89.9.3741

Cited by 158 publications

(107 citation statements)

References 44 publications

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“…A long-standing paradox concerning the regulation of CN is that the CN phosphatase activity towards pRII substrate was strongly inhibited by both CyPA-CsA and FKBP12-FK506, while the activity of CN towards pNPP was stimulated by approximately 4-fold in the presence of the drug complexes [28,29]. We confirmed these results for WT CN β.…”

Section: Further Evidence For the Multi-level Activation Of Cnsupporting

confidence: 79%

Cooperative autoinhibition and multi-level activation mechanisms of calcineurin

Wang

et al. 2016

Cell Res

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The Ca 2+ /calmodulin-dependent protein phosphatase calcineurin (CN), a heterodimer composed of a catalytic subunit A and an essential regulatory subunit B, plays critical functions in various cellular processes such as cardiac hypertrophy and T cell activation. It is the target of the most widely used immunosuppressants for transplantation, tacrolimus (FK506) and cyclosporin A. However, the structure of a large part of the CNA regulatory region remains to be determined, and there has been considerable debate concerning the regulation of CN activity. Here, we report the crystal structure of full-length CN (β isoform), which revealed a novel autoinhibitory segment (AIS) in addition to the well-known autoinhibitory domain (AID). The AIS nestles in a hydrophobic intersubunit groove, which overlaps the recognition site for substrates and immunosuppressant-immunophilin complexes. Indeed, disruption of this AIS interaction results in partial stimulation of CN activity. More importantly, our biochemical studies demonstrate that calmodulin does not remove AID from the active site, but only regulates the orientation of AID with respect to the catalytic core, causing incomplete activation of CN. Our findings challenge the current model for CN activation, and provide a better understanding of molecular mechanisms of CN activity regulation.

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Section: Further Evidence For the Multi-level Activation Of Cnsupporting

confidence: 79%

Cooperative autoinhibition and multi-level activation mechanisms of calcineurin

Wang

et al. 2016

Cell Res

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“…A Ca 2+ -dependent, CsA-inhibitory protein phosphatase activity was also detected Figure 4, pCyP B-CsA inhibited calcineurin activity with Ki of 4 9 0 nM. This value is comparable to the potency of the complexes formed by CsA and mammalian CyPs (Swanson et al, 1992;Bram et al, 1993). Neither CsA nor pCyP B alone inhibited calcineurin activity (data not shown).…”

Section: Complex Of Pcyp B-csa Inhibits Calcineurin Activitysupporting

confidence: 49%

“…Several cyclophilin-CsA complexes have been shown to inhibit the Ca 2+ calmodulin-dependent protein phosphatase calcineurin and to block T cell activation (Swanson et al, 1992;Bram et al, 1993). In an earlier article (Luan et al, 1993), we reported that CsA blocks a Ca 2+ -dependent signal transduction pathway in guard cells of fava bean.…”

Section: Complex Of Pcyp B-csa Inhibits Calcineurin Activitymentioning

confidence: 99%

pCyP B: a chloroplast-localized, heat shock-responsive cyclophilin from fava bean.

1994

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When the immunosuppressants cyclosporin A (CsA) and FK506 bind to their intracellular receptors (immunophilins), they form complexes that bind to calcineurin and block calcineurin-dependent signaling pathways in immune cells. Previously, we reported that higher plants also express immunophilins and have a Ca2+-dependent signaling pathway sensitive to immunophilin-ligand complexes. Based on an N-terminal peptide sequence of a chloroplast-localized cyclophilin (pCyP B), we isolated a cDNA clone encoding the preprotein of the cyclophilin. The deduced amino acid sequence of this cDNA starts with a putative transit sequence for chloroplast targeting. The mature pCyP B protein has rotamase activity with low-substrate specificity. Enzyme activity was inhibited by CsA with an inhibition constant of 3.9 nM. Similar to otherCyPs from mammalian cells, pCyP B, when complexed with CsA, inhibited the phosphatase activity of bovine calcineurin. The mRNA leve1 of pCyP B was high in leaf tissues but was not detectable in roots. Expression of the transcript in the leaf tissues was regulated by light and induced by heat shock. These findings illustrate the conserved nature of cyclophilin proteins among all of the eukaryotes and suggest that cyclophilins have a unique mode of regulation in higher plants.

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“…Ratiometric determinations were also made in cells treated with the Ca 2ϩ chelators BAPTA (Molecular Probes) and EDTA. Calcineurin Substrate Preparation-R II peptide was prepared and radiolabeled with [␥-32 P]ATP by cAMP-dependent protein kinase as described (25,26). Briefly, R II peptide (0.15 mM) was phosphorylated in 1 ml of phosphorylation reaction buffer (50 mM MOPS, pH 7.0, 2 mM MgSO 4 , 0.3 mM ATP, 2500 units of the catalytic subunit of cAMP-dependent protein kinase and 833 Ci/mmol of [␥-32 P]ATP) for 3 h at 30°C.…”

Section: ϩmentioning

confidence: 99%

Inhibition of Calcineurin Phosphatase Activity by a Calcineurin B Homologous Protein

Xiao¹,

Sikkink²,

Rusnak³

et al. 1999

Journal of Biological Chemistry

104

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Calcineurin, a Ca 2؉ /calmodulin-stimulated protein phosphatase, plays a key role in T-cell activation by regulating the activity of NFAT (nuclear factor of activated T cells), a family of transcription factors required for the synthesis of several cytokine genes. Calcineurin is the target of the immunosuppressive drugs cyclosporin A and FK506 complexed with their cytoplasmic receptors cyclophilin and FKBP12, respectively. In this study we report that calcineurin is also the target of a recently identified Ca 2؉ -binding protein, CHP (for calcineurin homologous protein), which shares a high degree of homology with the regulatory B subunit of calcineurin and with calmodulin. In Jurkat and HeLa cells, overexpression of CHP specifically impaired the nuclear translocation and transcriptional activity of NFAT but had no effect on AP-1 transcriptional activity and only a small (<25%) inhibitory effect on the transcriptional activity of NFB. Further study indicated that CHP inhibits calcineurin activity. In cells overexpressing CHP, the phosphatase activity of immunoprecipitated calcineurin was inhibited by ϳ50%; and in a reconstituted assay, the activity of purified calcineurin was inhibited up to 97% by the addition of purified recombinant CHP in a dose-dependent manner. Moreover, prolonged activation of Jurkat cells was associated with a decreased abundance of CHP, suggesting a possible regulatory mechanism allowing activation of calcineurin. CHP, therefore, is a previously unrecognized endogenous inhibitor of calcineurin activity.Stimulation of T cells results in the induction of several cytokine genes that are involved in the control of T-cell proliferation and immune responses (1-3). One of the signaling pathways mediating T-cell receptor activation involves the elevation of intracellular calcium ([Ca 2ϩ ] i ) and the activation of a key signaling enzyme, calcineurin (4, 5). A rise in [Ca 2ϩ ] i leads to the activation of calcineurin and the dephosphorylation and nuclear translocation of NFAT (nuclear factor of activated T cells).1 This in turn drives transcription of response genes such as the cytokines interleukin (IL)-2 and IL-4 and the CD44 ligand (6 -11). Calcineurin is a heterodimeric enzyme consisting of a catalytic A subunit (CnA) and a tightly associated Ca 2ϩ -binding regulatory B subunit (CnB) (12, 13). Maximal calcineurin phosphatase activity requires the Ca 2ϩ -dependent binding of calmodulin (CaM) to the CnA-CnB complex at a domain independent of the CnB-binding site on CnA (14).We recently identified a novel, ubiquitously expressed, Ca 2ϩ -binding protein, CHP (for calcineurin homologous protein), which shares a high degree of similarity with CnB (65%) and CaM (59%) (15). Although CHP was originally identified by screening a library to detect proteins that interacted with the Na ϩ /H ϩ exchanger isoform NHE1, subsequent studies indicated that CHP has actions that are independent of its regulation of NHE1. Overexpression of CHP in mutant NHE-deficient fibroblasts inhibits cell proliferation, ...

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Cyclosporin-mediated inhibition of bovine calcineurin by cyclophilins A and B.

Abstract: The

Cited by 158 publications

References 44 publications

Cooperative autoinhibition and multi-level activation mechanisms of calcineurin

Cooperative autoinhibition and multi-level activation mechanisms of calcineurin

pCyP B: a chloroplast-localized, heat shock-responsive cyclophilin from fava bean.

Inhibition of Calcineurin Phosphatase Activity by a Calcineurin B Homologous Protein

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