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. 1991 Dec 15;88(24):11431–11434. doi: 10.1073/pnas.88.24.11431

Characterization of a protein that regulates the DNA-binding activity of NF-AT, the nuclear factor of activated T cells.

A Granelli-Piperno 1, P McHugh 1
PMCID: PMC53149  PMID: 1722332

Abstract

NF-AT is a T-cell-restricted activity that regulates interleukin 2 expression by binding to positions -285 to -254 of the interleukin 2 gene. Prior studies have shown that NF-AT is found only in T cells that are appropriately stimulated with "two signals" and is sensitive to the immunosuppressive drugs cyclosporin A and FK506. However, NF-AT has yet to be characterized biochemically. Here we show that the activity of NF-AT in electrophoretic gel mobility-shift assays is greatly enhanced by a distinct protein with an apparent molecular mass of 28 kDa. This modulator has been enriched from the flow-through of NF-AT affinity columns and enhances by severalfold the activity of the factor that elutes from these columns. The modulator has a predominantly nuclear location, but unlike NF-AT, the modulator is found in nuclear extracts of many cell types, even unstimulated T cells and T cells immunosuppressed with cyclosporin A or FK506. This modulator protein may regulate NF-AT binding activity posttranscriptionally, and it should prove useful in the isolation and biochemical characterization of NF-AT.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abate C., Luk D., Curran T. A ubiquitous nuclear protein stimulates the DNA-binding activity of fos and jun indirectly. Cell Growth Differ. 1990 Oct;1(10):455–462. [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Brunvand M. W., Schmidt A., Siebenlist U. Nuclear factors interacting with the mitogen-responsive regulatory region of the interleukin-2 gene. J Biol Chem. 1988 Dec 15;263(35):18904–18910. [PubMed] [Google Scholar]
  4. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Durand D. B., Bush M. R., Morgan J. G., Weiss A., Crabtree G. R. A 275 basepair fragment at the 5' end of the interleukin 2 gene enhances expression from a heterologous promoter in response to signals from the T cell antigen receptor. J Exp Med. 1987 Feb 1;165(2):395–407. doi: 10.1084/jem.165.2.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Emmel E. A., Verweij C. L., Durand D. B., Higgins K. M., Lacy E., Crabtree G. R. Cyclosporin A specifically inhibits function of nuclear proteins involved in T cell activation. Science. 1989 Dec 22;246(4937):1617–1620. doi: 10.1126/science.2595372. [DOI] [PubMed] [Google Scholar]
  7. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Granelli-Piperno A., Inaba K., Steinman R. M. Stimulation of lymphokine release from T lymphoblasts. Requirement for mRNA synthesis and inhibition by cyclosporin A. J Exp Med. 1984 Dec 1;160(6):1792–1802. doi: 10.1084/jem.160.6.1792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Granelli-Piperno A., Nolan P., Inaba K., Steinman R. M. The effect of immunosuppressive agents on the induction of nuclear factors that bind to sites on the interleukin 2 promoter. J Exp Med. 1990 Dec 1;172(6):1869–1872. doi: 10.1084/jem.172.6.1869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Granelli-Piperno A., Nolan P. Nuclear transcription factors that bind to elements of the IL-2 promoter. Induction requirements in primary human T cells. J Immunol. 1991 Oct 15;147(8):2734–2739. [PubMed] [Google Scholar]
  11. Kadonaga J. T., Tjian R. Affinity purification of sequence-specific DNA binding proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5889–5893. doi: 10.1073/pnas.83.16.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Krönke M., Leonard W. J., Depper J. M., Arya S. K., Wong-Staal F., Gallo R. C., Waldmann T. A., Greene W. C. Cyclosporin A inhibits T-cell growth factor gene expression at the level of mRNA transcription. Proc Natl Acad Sci U S A. 1984 Aug;81(16):5214–5218. doi: 10.1073/pnas.81.16.5214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ohlsson H., Edlund T. Sequence-specific interactions of nuclear factors with the insulin gene enhancer. Cell. 1986 Apr 11;45(1):35–44. doi: 10.1016/0092-8674(86)90535-0. [DOI] [PubMed] [Google Scholar]
  14. Shaw J. P., Utz P. J., Durand D. B., Toole J. J., Emmel E. A., Crabtree G. R. Identification of a putative regulator of early T cell activation genes. Science. 1988 Jul 8;241(4862):202–205. doi: 10.1126/science.3260404. [DOI] [PubMed] [Google Scholar]
  15. Smith K. A. Interleukin-2: inception, impact, and implications. Science. 1988 May 27;240(4856):1169–1176. doi: 10.1126/science.3131876. [DOI] [PubMed] [Google Scholar]
  16. Ullman K. S., Northrop J. P., Verweij C. L., Crabtree G. R. Transmission of signals from the T lymphocyte antigen receptor to the genes responsible for cell proliferation and immune function: the missing link. Annu Rev Immunol. 1990;8:421–452. doi: 10.1146/annurev.iy.08.040190.002225. [DOI] [PubMed] [Google Scholar]
  17. Williams T. M., Eisenberg L., Burlein J. E., Norris C. A., Pancer S., Yao D., Burger S., Kamoun M., Kant J. A. Two regions within the human IL-2 gene promoter are important for inducible IL-2 expression. J Immunol. 1988 Jul 15;141(2):662–666. [PubMed] [Google Scholar]
  18. Wu C., Wilson S., Walker B., Dawid I., Paisley T., Zimarino V., Ueda H. Purification and properties of Drosophila heat shock activator protein. Science. 1987 Nov 27;238(4831):1247–1253. doi: 10.1126/science.3685975. [DOI] [PubMed] [Google Scholar]

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