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The T-cell transcription factor NFATp is a substrate for calcineurin and interacts with Fos and Jun
J Jain1, P G McCaffrey, Z Miner
1Division of Tumor Virology, Dana-Farber Cancer Institute, Boston, Massachusetts.
Abstract:
Transcription of lymphokine genes in activated T cells is inhibited by the immunosuppressive agents cyclosporin A and FK506, which act by blocking the phosphatase activity of calcineurin. NFAT, a DNA-binding protein required for interleukin-2 gene transcription, is a potential target for calcineurin, cyclosporin A and FK506. NFAT contains a subunit (NFATp) which is present in unstimulated T cells and which forms a complex with Fos and Jun proteins in the nucleus of activated T cells. Here we report that NFATp is a DNA-binding phosphoprotein of relative molecular mass approximately 120,000 and is a substrate for calcineurin in vitro. Purified NFATp forms DNA-protein complexes with recombinant Jun homodimers or Jun-Fos heterodimers; the DNA-binding domains of Fos and Jun are essential for the formation of the NFATp-Fos-Jun-DNA complex. The interaction between the lymphoid-specific factor NFATp and the ubiquitous transcription factors Fos and Jun provides a novel mechanism for combinatorial regulation of interleukin-2 gene transcription, which integrates the calcium-dependent and the protein-kinase C-dependent pathways of T-cell activation.
Insights
Cyclosporin A and FK506 inhibit T cell activation by blocking calcineurin phosphatase activity. This study identifies NFATp as a calcineurin substrate, crucial for interleukin-2 gene transcription via interaction with Fos and Jun proteins.
Area of Science:
- Immunology
- Molecular Biology
- Cellular Biology
Background:
- Immunosuppressive agents cyclosporin A and FK506 inhibit T cell activation by targeting calcineurin.
- Calcineurin's phosphatase activity is critical for lymphokine gene transcription in T cells.
- NFAT (Nuclear Factor of Activated T cells) is a key DNA-binding protein involved in interleukin-2 gene transcription.
Purpose of the Study:
- To investigate the role of NFATp, a subunit of NFAT, in T cell activation.
- To determine if NFATp is a substrate for calcineurin.
- To elucidate the mechanism by which NFATp interacts with Fos and Jun proteins.
Main Methods:
- In vitro biochemical assays to test calcineurin's activity on NFATp.
- Purification of NFATp.
- Analysis of DNA-protein complex formation using recombinant Jun and Fos proteins.
Main Results:
- NFATp was identified as a DNA-binding phosphoprotein (approx. 120,000 Da) and a direct substrate for calcineurin in vitro.
- Purified NFATp formed DNA-protein complexes with Jun homodimers and Jun-Fos heterodimers.
- The DNA-binding domains of Fos and Jun were essential for the formation of the NFATp-Fos-Jun-DNA complex.
Conclusions:
- NFATp is a critical component in the regulation of interleukin-2 gene transcription.
- The interaction between NFATp and Fos/Jun provides a novel mechanism for combinatorial gene regulation.
- This interaction integrates calcium-dependent and protein-kinase C-dependent signaling pathways in T cell activation.