title: Phosphorylation of the transcription factor NFATp inhibits its DNA binding activity in cyclosporin A-treated human B and T cells.
abstract: Cyclosporin A (CsA) exerts its immunosuppressive effect by inhibiting the activity of nuclear factor of activated T cells (NFAT), thus preventing transcriptional induction of several cytokine genes. This effect is thought to be largely mediated through inactivation of the phosphatase calcineurin, which in turn inhibits translocation of an NFAT component to the nucleus. Here we report that CsA treatment of Raji B and Jurkat T cell lines yields a phosphorylated form of NFATp that is inhibited in DNA-binding and in its ability to form an NFAT complex with Fos and Jun. Immunoblot analyses and metabolic labeling with [32P]orthophosphate show that CsA alters NFATp migration on SDS-polyacrylamide gel electrophoresis by increasing its phosphorylation level without affecting subcellular distribution. Dephosphorylation by in vitro treatment with calcineurin or alkaline phosphatase restores NFATp DNA binding activity and its ability to reconstitute an NFAT complex with Fos and Jun proteins. These data point to a new mechanism for CsA-sensitive regulation of NFATp in which dephosphorylation is critical for DNA binding.
authors.affiliation: Department of Pathology, Roger Williams Medical Center-Brown University, Providence, Rhode Island 02908, USA.
authors.forename: J
authors.initials: J
authors.lastname: Park
authors.forename: N R
authors.initials: NR
authors.lastname: Yaseen
authors.forename: P G
authors.initials: PG
authors.lastname: Hogan
authors.forename: A
authors.initials: A
authors.lastname: Rao
authors.forename: S
authors.initials: S
authors.lastname: Sharma
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology*; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007375:Interleukin-1 / Q000235:genetics; D007422:Intestines / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D002148:Calmodulin-Binding Proteins; D004268:DNA-Binding Proteins; D007375:Interleukin-1; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D010710:Phosphates; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors; D016572:Cyclosporine; D000469:Alkaline Phosphatase; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases