Inhibition of NFATx activation by an oligopeptide: disrupting the interaction of NFATx with calcineurin.
Calcium-dependent phosphatase calcineurin (CN) regulates the activation and nuclear translocation of NFAT. We identify here a novel CN-binding motif in one member of the NFAT family, NFATx, and a peptide based on this motif, Pep3. Pep3 binds CN and competes with wild-type NFATx for CN interaction. Amino acid mutations within Pep3 show that multiple amino acid residues are required for the effective functions of Pep3. Ectopic expression of Pep3 in a Th clone via a retrovirus-mediated gene transfer could selectively block the nuclear translocation of endogenous NFATx, whereas it had little effect on the nuclear translocation of another member of the NFAT family, NFATp. Furthermore, in transfection experiments, Pep3 also blocked the nuclear translocation of transfected NFATx, but not NFATp, in the B cell line M12, demonstrating specific inhibition of Pep3 for NFATx. Importantly, several cytokines produced by the T cell clone were severely repressed by ectopic Pep3, and indeed, the production of these cytokines was enhanced by the expression of wild-type NFATx. Our results show selective inhibition of NFATx activation and cytokine expression by Pep3 and suggest a new approach for studying the biology of each NFAT family member. This approach may provide an opportunity for pharmacological targeting of Ca(2+)-dependent signaling events.
Liu
J
J
Department of Immunology, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304, USA.
Arai
K
K
Arai
N
N
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019703:Calcineurin / Q000378:metabolism*; D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D009842:Oligopeptides / Q000235:genetics / Q000494:pharmacology*; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D016207:Cytokines; D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; C494008:NFATC3 protein, human; C494009:Nfatc3 protein, mouse; D009687:Nuclear Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; D014157:Transcription Factors; D019703:Calcineurin