A conserved docking surface on calcineurin mediates interaction with substrates and immunosuppressants.
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.
Rodríguez
Antonio
A
Department of Vascular Biology and Inflammation, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain. a.rodriguez@uam.es
Roy
Jagoree
J
Martínez-Martínez
Sara
S
López-Maderuelo
María Dolores
MD
Niño-Moreno
Perla
P
Ortí
Leticia
L
Pantoja-Uceda
David
D
Pineda-Lucena
Antonio
A
Cyert
Martha S
MS
Redondo
Juan Miguel
JM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019703:Calcineurin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D003001:Cloning, Molecular; D003198:Computer Simulation; D017124:Conserved Sequence; D017930:Genes, Reporter; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D020104:Immunophilins / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000378:metabolism* / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D019169:Jurkat Cells; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D050778:NFATC Transcription Factors / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D015398:Signal Transduction; D013499:Surface Properties; D022061:Tacrolimus Binding Protein 1A / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D065095:Calcineurin Inhibitors; D007166:Immunosuppressive Agents; D047908:Intracellular Signaling Peptides and Proteins; D050778:NFATC Transcription Factors; D010455:Peptides; C486302:RCN1 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C070393:CNA1 protein, S cerevisiae; D019703:Calcineurin; C525673:PPP3CA protein, human; C525676:PPP3R1 protein, human; D022061:Tacrolimus Binding Protein 1A; D020104:Immunophilins