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Identification of a physical interaction between calcineurin and nuclear factor of activated T cells (NFATp)
S Wesselborg1, D A Fruman, J K Sagoo
1Division of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Abstract:
In T lymphocytes, the calcium/calmodulin-dependent serine/threonine phosphatase, calcineurin, plays a pivotal role in transducing membrane-associated signals to the nucleus. One of the putative targets of calcineurin is the pre-existing, cytosolic component of the nuclear factor of activated T cells (NFATp; also referred to as NFAT1), which is one of several transcription factors required for the expression of interleukin 2. Inhibition of calcineurin by the immunosuppressive drugs cyclosporin A and FK506 prevents dephosphorylation of NFATp and its translocation to the nucleus. However, a physical interaction between calcineurin and NFATp has not been demonstrated. Here we demonstrate the binding of NFATp from lysates of T cells to immobilized calcineurin. Stimulation of T cells with calcium ionophore induced a shift in the molecular weight of NFATp that is due to its dephosphorylation. This dephosphorylation was inhibited by treatment of T cells with cyclosporin A or FK506 prior to stimulation. Of note, both the phosphorylated and the dephosphorylated form of NFATp bound to calcineurin. Furthermore, the binding of both forms of NFATp to calcineurin was inhibited by pretreatment of calcineurin with a complex of FK506 and its ligand FKBP12. Taken together these data strongly suggest a direct interaction of calcineurin with NFATp and that this interaction does not depend upon the phosphorylation sites of NFATp affected by activation.
Insights
Calcineurin directly binds to nuclear factor of activated T cells (NFATp), a key transcription factor in T lymphocytes. This interaction is crucial for immune signaling and is unaffected by NFATp phosphorylation status.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- Calcineurin is a critical phosphatase in T lymphocyte signaling, mediating nuclear translocation of transcription factors.
- Nuclear Factor of Activated T cells (NFATp) is a cytosolic protein essential for interleukin-2 gene expression.
- The physical interaction between calcineurin and NFATp has remained unconfirmed despite functional evidence.
Purpose of the Study:
- To investigate and demonstrate a direct physical interaction between calcineurin and NFATp.
- To determine if NFATp phosphorylation state influences its binding to calcineurin.
- To explore the impact of immunosuppressive drugs on the calcineurin-NFATp interaction.
Main Methods:
- Affinity binding assays using immobilized calcineurin and T cell lysates.
- Analysis of NFATp molecular weight shifts indicative of dephosphorylation upon T cell stimulation.
- Assessment of immunosuppressive drug effects (cyclosporin A, FK506) on NFATp dephosphorylation and binding.
- Inhibition studies using FK506-FKBP12 complex to probe binding mechanisms.
Main Results:
- NFATp was shown to bind to immobilized calcineurin in T cell lysates.
- T cell stimulation induced NFATp dephosphorylation, confirmed by molecular weight shift.
- This dephosphorylation was blocked by prior treatment with cyclosporin A or FK506.
- Both phosphorylated and dephosphorylated NFATp forms bound to calcineurin.
- Binding of NFATp to calcineurin was inhibited by FK506-FKBP12 complex.
Conclusions:
- Direct physical interaction between calcineurin and NFATp is demonstrated.
- The interaction is independent of the specific phosphorylation sites on NFATp regulated during T cell activation.
- These findings provide a molecular basis for calcineurin's role in T cell activation pathways.