IDENTIFICATION OF A PHYSICAL INTERACTION BETWEEN CALCINEURIN AND NUCLEAR FACTOR OF ACTIVATED T-CELLS (NFATP)

In T lymphocytes, the calcium/calmodulin-dependent serine/threonine ph osphatase, calcineurin, plays a pivotal role in transducing membrane-a ssociated signals to the nucleus. One of the putative targets of calci neurin 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 interl eukin 2. Inhibition of calcineurin by the immunosuppressive drugs cycl o-sporin A and FK506 prevents dephosphorylation of NFATp and its trans location to the nucleus. However, a physical interaction between calci neurin and NFATp has not been demonstrated. Here we demonstrate the bi nding of NFATp from lysates of T cells to immobilized calcineurin. Sti mulation of T cells with calcium ionophore induced a shift in the mole cular weight of NFATp that is due to its dephosphorylation. This depho sphorylation was inhibited by treatment of T cells with cyclo-sporin A or FK506 prior to stimulation. Of note, both the phosphorylated and t he dephosphorylated form of NFATp bound to calcineurin. Furthermore, t he binding of both forms of NFATp to calcineurin was inhibited by pret reatment of calcineurin with a complex of FK506 and its ligand FKBP12. Taken together these data strongly suggest a direct interaction of ca lcineurin with NFATp and that this interaction does not depend upon th e phosphorylation sites of NFATp affected by activation.