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Isolation of the cyclosporin-sensitive T cell transcription factor NFATp
P G McCaffrey1, C Luo, T K Kerppola
1Division of Tumor Virology, Dana-Farber Cancer Institute, Boston, MA.
Abstract:
Nuclear factor of activated T cells (NFAT) is a transcription factor that regulates expression of the cytokine interleukin-2 (IL-2) in activated T cells. The DNA-binding specificity of NFAT is conferred by NFATp, a phosphoprotein that is a target for the immunosuppressive compounds cyclosporin A and FK506. Here, the purification of NFATp from murine T cells and the isolation of a complementary DNA clone encoding NFATp are reported. A truncated form of NFATp, expressed as a recombinant protein in bacteria, binds specifically to the NFAT site of the murine IL-2 promoter and forms a transcriptionally active complex with recombinant protein fragment react with T cell NFATp. The molecular cloning of NFATp should allow detailed analysis of a T cell transcription factor that is central to initiation of the immune response.
Insights
Researchers purified Nuclear Factor of Activated T cells p (NFATp) from T cells and cloned its DNA. This transcription factor is crucial for initiating immune responses by regulating interleukin-2 (IL-2) gene expression.
Area of Science:
- Immunology
- Molecular Biology
- Transcription Factor Research
Background:
- Nuclear factor of activated T cells (NFAT) regulates interleukin-2 (IL-2) expression in T cells.
- NFATp, a phosphoprotein, dictates DNA-binding specificity and is targeted by immunosuppressants like cyclosporin A and FK506.
Purpose of the Study:
- To purify NFATp from murine T cells.
- To isolate a complementary DNA (cDNA) clone encoding NFATp.
- To analyze the function of NFATp in T cell transcription.
Main Methods:
- Purification of NFATp from murine T cells.
- Isolation of NFATp-encoding cDNA.
- Expression of a truncated NFATp as a recombinant protein in bacteria.
- Electrophoretic mobility shift assays (EMSAs) to assess DNA-binding specificity.
Main Results:
- NFATp was successfully purified from murine T cells.
- A cDNA clone encoding NFATp was isolated.
- Recombinant truncated NFATp specifically bound to the NFAT site on the murine IL-2 promoter.
- Recombinant NFATp formed a transcriptionally active complex.
Conclusions:
- The molecular cloning of NFATp enables detailed analysis of this key T cell transcription factor.
- NFATp plays a central role in initiating the immune response.
- Understanding NFATp function is critical for T cell activation and regulation.