Real-Time Monitoring of Calcineurin Activity in Living Cells: Evidence for Two Distinct Ca-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 CaCl2 caused an increase in calcineurin activity with an initial peak, and then approached a sustained constant level in a concentration-dependent manner. In CaCl2-sensitive mutants such as Δpmc1, the response was markedly enhanced reflecting its high intracellular Ca. Agents expected to induce Ca 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 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. The Pck2 protein kinase C-Pmk1 MAP kinase pathway was required for the stimulation of calcineurin via Yam8/Cch1 mediated Ca influx, but it was not required for the stimulation by elevated extracellular Ca, suggesting two distinct pathways for calcineurin activation.