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Direct demonstration of NFATp dephosphorylation and nuclear localization in activated HT-2 cells using a specific
1Department of Cell Biology and Inflammation Research, Upjohn Company, Kalamazoo, Michigan 49001, USA.
Abstract:
Nuclear factor of activated T cells (NFAT) regulates transcription of a number of cytokine genes, and NFAT DNA binding activity is stimulated following T cell activation. Several lines of evidence have suggested that NFAT is a substrate for calcineurin, a serine/threonine phosphatase. Using a polyclonal antibody to murine NFATp, Western blot analysis of various mouse tissues demonstrated that the 110-130-kDa NFATp protein was highly expressed in thymus and spleen. Treatment of immunoprecipitated NFATp from untreated HT-2 cells with calcineurin resulted in the dephosphorylation of NFATp, demonstrating that NFATp is an in vitro substrate for calcineurin. NFATp immunoprecipitated from 32P-labeled HT-2 cells migrated as an approximately 120-kDa protein that was localized to the cytosol of the cells. Treatment of the cells with ionomycin resulted in a decrease in the molecular weight of NFATp and a loss of 32P, consistent with NFATp dephosphorylation. The dephosphorylation of NFATp was accompanied by localization of the protein to the nuclear fraction. Both of these events were blocked by preincubation of the cells with FK506, a calcineurin inhibitor, consistent with the hypothesis that NFATp is a calcineurin substrate in cells.
Insights
Nuclear factor of activated T cells (NFAT) dephosphorylation by calcineurin is crucial for T cell activation. This study shows NFATp is a calcineurin substrate, moving to the nucleus after T cell stimulation.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Nuclear factor of activated T cells (NFAT) controls cytokine gene transcription.
- NFAT DNA binding activity increases upon T cell activation.
- Evidence suggests NFAT is a substrate for calcineurin, a phosphatase.
Purpose of the Study:
- To investigate the relationship between NFAT and calcineurin.
- To confirm NFATp as a calcineurin substrate.
- To elucidate the role of calcineurin in NFAT regulation.
Main Methods:
- Western blot analysis of NFATp expression in mouse tissues.
- In vitro dephosphorylation assays using immunoprecipitated NFATp and calcineurin.
- Analysis of NFATp phosphorylation and cellular localization in HT-2 cells treated with ionomycin and FK506.
Main Results:
- NFATp (110-130 kDa) highly expressed in thymus and spleen.
- Calcineurin dephosphorylated NFATp in vitro.
- Ionomycin treatment caused NFATp dephosphorylation and nuclear translocation.
- FK506 blocked these ionomycin-induced events, confirming calcineurin's role.
Conclusions:
- NFATp is a direct in vitro substrate for calcineurin.
- Calcineurin-mediated dephosphorylation of NFATp is essential for its nuclear translocation.
- This process is critical for T cell activation and cytokine gene regulation.