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Vol. 17, Issue 11, 4790-4800, November 2006

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Real-Time Monitoring of Calcineurin Activity in Living Cells: Evidence for Two Distinct Ca²⁺-dependent Pathways in Fission Yeast

Lu Deng^*, Reiko Sugiura^*,, Mai Takeuchi^*, Masahiro Suzuki^*, Hidemine Ebina^*, Tomonori Takami^*, Atsushi Koike^*, Shiori Iba^*, and Takayoshi Kuno^*

*Division of Molecular Pharmacology and Pharmacogenomics, Department of Genome Sciences, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan; and Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Higashi-Osaka 577-8502, Japan

Submitted June 19, 2006; Accepted August 15, 2006
Monitoring Editor: Fred Chang

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 pmc1, the response was markedly enhanced, reflecting its high intracellular Ca²⁺. Agents expected to induce Ca²⁺ 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 Ca²⁺ 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 Ca²⁺. The Pck2 protein kinase C-Pmk1 mitogen-activate protein kinase pathway was required for the stimulation of calcineurin via Yam8/Cch1-mediated Ca²⁺ influx, but it was not required for the stimulation by elevated extracellular Ca²⁺, suggesting two distinct pathways for calcineurin activation.

Address correspondence to: Takayoshi Kuno (tkuno{at}med.kobe-u.ac.jp)

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