The immunosuppressive drugs cyclosporin A and tacrolimus inhibit membrane depolarization-induced CREB transcriptional activity at the coactivator level

1 Cyclosporin A and tacrolimus are clinically important immunosuppressive drugs directly targeting the transcription factor nuclear factor of activated T cells ( NFAT). Through inhibition of calcineurin phosphatase activity they block the dephosphorylation and thus activation of NFAT. Cyclosporin A and tacrolimus also inhibit other calcineurin-dependent transcription factors including the ubiquitously expressed cAMP response element-binding protein ( CREB). Membrane depolarization by phosphorylating CREB on Ser119 leads to the recruitment of its coactivator CREB-binding protein (CBP) that stimulates initiation of transcription. 2 It was unknown at what step in CREB-mediated transcription cyclosporin A and tacrolimus interfere. 3 In transient transfection experiments, using GAL4-CREB fusion proteins and a pancreatic islet beta-cell line, cyclosporin A inhibited depolarization-induced activation of CREB proteins which carried various deletions or mutations throughout their sequence providing no evidence for the existence of a distinct CREB domain conferring cyclosporin A sensitivity. In a mammalian two-hybrid assay, cyclosporin A did not inhibit Ser119-dependent interaction of CREB with its coactivator CBP. 4 Using GAL4-CBP fusion proteins, cyclosporin A inhibited depolarization-induced CBP activity, with cyclosporin A-sensitive domains mapped to both the N- ( aa 1 - 451) and C-terminal ( aa 2040 2305) ends of CBP. The depolarization-induced transcriptional activity of the CBP C-terminus was enhanced by overexpression of calcineurin and was inhibited by cyclosporin A and tacrolimus in a concentration-dependent manner with IC50 values ( 10 and 1 nM, respectively) consistent with their known IC50 values for inhibition of calcineurin. 5 These data suggest that, in contrast to NFAT, cyclosporin A and tacrolimus inhibit CREB transcriptional activity at the coactivator level.