Identification of an inducible regulator of c-myb expression during T-cell activation.
Resting T cells express very low levels of c-Myb protein. During T-cell activation, c-myb expression is induced and much of the increase in expression occurs at the transcriptional level. We identified a region of the c-myb 5' flanking sequence that increased c-myb expression during T-cell activation. In vivo footprinting by ligation-mediated PCR was performed to correlate in vivo protein binding with functional activity. A protein footprint was visible over this region of the c-myb 5' flanking sequence in activated T cells but not in unactivated T cells. An electrophoretic mobility shift assay (EMSA) with nuclear extract from activated T cells and an oligonucleotide of this binding site demonstrated a new protein-DNA complex, referred to as CMAT for c-myb in activated T cells; this complex was not present in unactivated T cells. Because the binding site showed some sequence similarity with the nuclear factor of activated T cells (NFAT) binding site, we compared the kinetics of induction of the two binding complexes and the molecular masses of the two proteins. Studies of the kinetics of induction showed that the NFAT EMSA binding complex appeared earlier than the CMAT complex. The NFAT protein migrated more slowly in a sodium dodecyl sulfate-polyacrylamide gel than the CMAT protein did. In addition, an antibody against NFAT did not cross-react with the CMAT protein. The appearance of the CMAT binding complex was inhibited by both cyclosporin A and rapamycin. The CMAT protein appears to be a novel inducible protein involved in the regulation of c-myb expression during T-cell activation.
Phan
S C
SC
Center for Molecular Biology in Medicine, Palo Alto VAMC, California 94304, USA.
Feeley
B
B
Withers
D
D
Boxer
L M
LM
D001483:Base Sequence; D002453:Cell Cycle; D002460:Cell Line; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D009857:Oncogenes; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D020598:Proto-Oncogene Proteins c-myb; D011519:Proto-Oncogenes; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D015534:Trans-Activators / Q000096:biosynthesis* / Q000302:isolation & purification; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D011994:Recombinant Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D008156:Luciferases