Calcineurin is a phosphatase that targets the transcription factor， nuclear factor of activated T-cells (NFAT) dephosphorylates multiple sites along NFAT's regulatory domain. The calcineurin-NFAT complex interaction is mediated through two conserved binding motifs known as the PxIxIT and LxVP， which are located at the N- and C- terminus to the phosphorylation sites. The vast range of cellular processes regulated by the calcineurin-NFAT interaction has aroused great interest in the investigation of the structural aspects that govern their complex formation and in the discovery of protein-protein interaction inhibitors; the latter interfere with calcineurin-NFAT complex formation while keeping calcineurin's c... More
Calcineurin is a phosphatase that targets the transcription factor， nuclear factor of activated T-cells (NFAT) dephosphorylates multiple sites along NFAT's regulatory domain. The calcineurin-NFAT complex interaction is mediated through two conserved binding motifs known as the PxIxIT and LxVP， which are located at the N- and C- terminus to the phosphorylation sites. The vast range of cellular processes regulated by the calcineurin-NFAT interaction has aroused great interest in the investigation of the structural aspects that govern their complex formation and in the discovery of protein-protein interaction inhibitors; the latter interfere with calcineurin-NFAT complex formation while keeping calcineurin's catalytic site free. To assist additional biophysical study of the calcineurin-NFAT structure-function relation and to screen for new inhibitors， we present a robust and cost-effective Enzyme Linked Immuno Sorbent Assay (ELISA) that is based on the interaction of calcineurin with the NFAT homology region. The latter includes the two calcineurin's binding sites， in addition to the phosphorylation sites. The ELISA experiment shown here can thus be applied towards the study of important structural aspects of the complex and for the discovery of new inhibitors. This will allow for a better understanding of T-cell activation switch.
