Real-time monitoring of calcineurin activity in living cells: evidence for two distinct Ca2+-dependent pathways in fission yeast.

In fission yeast, calcineurin dephosphorylates and activates the Prz1 transcription factor. Here, we identified the calcineurin-dependent response element (CDRE) in the promoter region of prz1(+) gene and monitored the calcineurin activity in living cells using a destabilized luciferase reporter gene fused to three tandem repeats of CDRE. Elevated extracellular CaCl(2) caused an increase in calcineurin activity with an initial peak and then approached a sustained constant level in a concentration-dependent manner. In CaCl(2)-sensitive mutants such as Deltapmc1, the response was markedly enhanced, reflecting its high intracellular Ca(2+). Agents expected to induce Ca(2+) influx showed distinct patterns of the CDRE-reporter activity, suggesting different mechanisms of calcineurin activation. Knockout of yam8(+) or cch1(+) encoding putative subunits of a Ca(2+) channel abolished the activation of calcineurin upon exposure to various stimuli, including high extracellular NaCl and cell wall-damaging agents. However, knockout of yam8(+) or cch1(+) did not affect the activation of calcineurin upon stimulation by elevated extracellular Ca(2+). The Pck2 protein kinase C-Pmk1 mitogen-activate protein kinase pathway was required for the stimulation of calcineurin via Yam8/Cch1-mediated Ca(2+) influx, but it was not required for the stimulation by elevated extracellular Ca(2+), suggesting two distinct pathways for calcineurin activation.