Characterization of NF(P), the nuclear factor that interacts with the regulatory P sequence (5'-CGAAAATTTCC-3') of the human interleukin-4 gene: relationship to NF-kappa B and NF-AT

Biochem Biophys Res Commun. 1994 Mar 15;199(2):439-46. doi: 10.1006/bbrc.1994.1248.

Abstract

The P sequence of the human interleukin-4 (IL-4) gene, which was defined as a responsive element for phorbol 12-myristate 13-acetate and calcium ionophore (A23187) in Jurkat T cells, shares sequence similarity with the NF-kappa B and the NF-AT binding sites. We examined whether NF(P), a nuclear factor specific for the P sequence, is related to NF-kappa B and NF-AT. NF-kappa B (P65 or P65/P50 heterodimer) bound to the P sequence in electrophoretic mobility shift assays (EMSA) and activated transcription through the P sequence when expression plasmids were cotransfected with P sequence-driven reporter plasmids in Jurkat T cells. In EMSAs, NF(P) binding was inhibited by the unlabeled NF-AT binding site but not by the unlabeled AP1 binding site and purified NF-AT contained an activity that bound to the P sequence. Both mobility shift and sequence specificity of NF-AT were similar to those of NF(P) and only a small amount of P65 was detected in NF(P) in crude nuclear extracts. These results indicate that the component(s) of NF-AT has the potential to reconstitute NF(P) whereas NF-kappa B alone cannot account for NF(P) in crude extracts. Unlike NF-AT, NF(P) does not contain AP1 as its DNA binding component.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Binding Sites
  • Calcimycin / pharmacology
  • Cell Line
  • DNA / metabolism*
  • DNA-Binding Proteins / isolation & purification
  • DNA-Binding Proteins / metabolism*
  • Humans
  • Interleukin-4 / genetics*
  • Luciferases / biosynthesis
  • Luciferases / metabolism
  • Molecular Sequence Data
  • NF-kappa B / metabolism*
  • NFATC Transcription Factors
  • Nuclear Proteins / isolation & purification
  • Nuclear Proteins / metabolism*
  • Oligodeoxyribonucleotides / chemical synthesis
  • Oligodeoxyribonucleotides / metabolism*
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Regulatory Sequences, Nucleic Acid*
  • Substrate Specificity
  • T-Lymphocytes
  • Tetradecanoylphorbol Acetate / pharmacology
  • Transcription Factors / isolation & purification
  • Transcription Factors / metabolism*
  • Transfection
  • Tumor Cells, Cultured

Substances

  • DNA-Binding Proteins
  • NF-kappa B
  • NFATC Transcription Factors
  • Nuclear Proteins
  • Oligodeoxyribonucleotides
  • Recombinant Proteins
  • Transcription Factors
  • Interleukin-4
  • Calcimycin
  • DNA
  • Luciferases
  • Tetradecanoylphorbol Acetate