The major goal of this proposal is to gain a molecular understanding of the mechanism of signal transduction by calcineurin during activation of T lymphocytes. The investigator has identified calcineurin as an important enzyme in the T cell receptor (TCR)-mediated signal transduction pathway leading to interleukin-2 (IL-2) production and as a common target for the immunosuppressive drugs cyclosporin A and FK506. Calcineurin is known to modulate the activities of at least three distinct classes of transcription factor including NF-AT, NF-kB and AP-1. However, many of the signaling molecules involved in the activation of NF-kB and AP-1 by calcineurin are not known. Calcineurin exists in multiple isoforms; it has been assumed that the a isoform of calcineurin mediates TCR signaling. Recently, calcineurin a was knocked out in mice, but no defect in the TCR-mediated IL-2 production was observed. In his preliminary studies, he has found that another isoform, calcineurin b, is more abundant than calcineurin a in T cells, suggesting that calcineurin b may play a dominant role in TCR signaling. Using FK506-resistant calcineurin mutants, he proposes to assess whether calcineurin b is sufficient to mediate TCR signaling. He will apply the yeast two-hybrid system to identify the substrates and associated proteins for calcineurin b to uncover signaling molecules involved in the activation of NF-kB and AP-1 by calcineurin. Alternatively, he will also employ catalytically inactive calcineurin mutants to identify calcineurin substrates from Jurkat cell extracts in vitro. Using partial cDNA sequences obtained from the yeast two-hybrid screen or oligopeptide sequences obtained from in vitro binding, the full length cDNA encoding putative calcineurin substrates and associated proteins will be cloned and further characterized in the context of TCR signaling. The identification of new signaling molecules through these studies will not only shed light on the molecular mechanism of TR signal transduction, but will also offer new targets for designing more specific and less toxic immunosuppressants.
