A central role for Fos in human B- and T-cell NFAT (nuclear factor of activated T cells): an acidic region is required for in vitro assembly.
Nuclear factor of activated T cells (NFAT) is a multicomponent transcription factor that contains Fos and Jun family proteins in addition to a constitutively expressed factor(s). It is important for the production of interleukin 2 (IL-2) by T cells and is also expressed in B cells. Here we show that NFAT complexes in B- and T-cell nuclear extracts can be supershifted prominently with Fos antibodies and to a variable extent with Jun family protein antibodies. Fos and Jun proteins appear to participate in NFAT complexes as heterodimers, since efficient in vitro reconstitution of NFAT in unstimulated B- or T-cell nuclear extracts required both Fos and Jun. Using Fos and Jun deletion derivatives, we found that an acidic Fos region (amino acids 118 to 138), outside the DNA binding and dimerization domains, was necessary for the in vitro reconstitution of the NFAT complex in both B- and T-lymphocyte extracts although it was not required for binding to an AP-1 site. Fos-Jun heterodimers exhibited low-affinity direct binding to the NFAT site in the absence of nuclear extracts. This binding also required the Fos acidic region, amino acids 118 to 138. Mutating a variant AP-1 site in the NFAT oligonucleotide abolished both direct binding of Fos-Jun heterodimers and in vitro reconstitution of NFAT. These results demonstrate a central role of Fos in NFAT complex formation in both B and T lymphocytes and show that NFAT assembly involves direct binding of Fos-Jun heterodimers to a variant AP-1 site within the human NFAT recognition site.
Yaseen
N R
NR
Department of Pathology, Roger Williams Cancer/Medical Center, Brown University, Providence, Rhode Island 02908.
Park
J
J
Kerppola
T
T
Curran
T
T
Sharma
S
S
D001402:B-Lymphocytes / Q000737:chemistry*; D001483:Base Sequence; D002467:Cell Nucleus / Q000737:chemistry; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006801:Humans; D008213:Lymphocyte Activation; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011485:Protein Binding; D011487:Protein Conformation; D016760:Proto-Oncogene Proteins c-fos / Q000032:analysis* / Q000235:genetics; D016755:Proto-Oncogene Proteins c-jun / Q000032:analysis* / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D013601:T-Lymphocytes / Q000737:chemistry*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry*; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D014157:Transcription Factors