A Conserved Docking Surface on Calcineurin Mediates Interaction with Substrates and Immunosuppressants
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abstract
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The phosphatase calcineurin, a target of the immunosuppressants cyclosporin A and FK506, dephosphorylates NFAT transcription factors to promote immune activation and development of the vascular and nervous systems. NFAT interacts with calcineurin through distinct binding motifs: the PxIxIT and LxVP sites. Although many calcineurin substrates contain PxIxIT motifs, the generality of LxVP-mediated interactions is unclear. We define critical residues in the LxVP motif, and we demonstrate its binding to a hydrophobic pocket at the interface of the two calcineurin subunits. Mutations in this region disrupt binding of mammalian calcineurin to NFATC1 and the interaction of yeast calcineurin with substrates including Rcn1, which contains an LxVP motif. These mutations also interfere with calcineurin-immunosuppressant binding, and an LxVP-based peptide competes with immunosuppressant-immunophilin complexes for binding to calcineurin. These studies suggest that LxVP-type sites are a common feature of calcineurin substrates, and that immunosuppressant-immunophilin complexes inhibit calcineurin by interfering with this mode of substrate recognition. © 2009 Elsevier Inc. All rights reserved.
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funding provided via
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Centro de Investigación Biomédica en Red Enfermedades Cardiovasculares, CIBERCV: RD06/0014/005 Grant
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Comunidad de Madrid: CAM-S2006/BIO-0194 Grant
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European Commission, EC: EICOSANOX LSHM-CT-2004-0050033 Grant
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Ministerio de Ciencia e Innovación, MICINN Grant
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Ministerio de Educación Superior, Ciencia y Tecnología, República Dominicana, MESCYT Grant
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Ministerio de Sanidad, Consumo y Bienestar, MSCBS Grant
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Mountain Equipment Co-operative, MEC: SAF2006-08348 Grant
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National Institute of General Medical Sciences, NIGMS: R01GM048729 Grant
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National Institutes of Health, NIH: GM-48728 Grant
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PI060122, S2006/BIO-0236 Grant
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Research
keywords
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HUMDISEASE; MOLIMMUNO; SIGNALING calcineurin; cyclosporin A; immunophilin; tacrolimus; article; circular dichroism; controlled study; enzyme activity; HeLa cell; human; human cell; in vitro study; leukemia cell line; nonhuman; point mutation; protein expression; yeast; Amino Acid Motifs; Amino Acid Sequence; Animals; Binding Sites; Calcineurin; Cloning, Molecular; Computer Simulation; Conserved Sequence; Genes, Reporter; Humans; Hydrophobicity; Immunophilins; Immunosuppressive Agents; Jurkat Cells; Mice; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; NFATC Transcription Factors; Peptides; Protein Conformation; Recombinant Fusion Proteins; Saccharomyces cerevisiae Proteins; Signal Transduction; Surface Properties; Tacrolimus Binding Protein 1A; Transcription, Genetic; Transfection; Mammalia
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