| 16928959 |
Deng L, Sugiura R, Takeuchi M, Suzuki M, Ebina H, Takami T, Koike A, Iba S, Kuno T: Real-time monitoring of calcineurin activity in living cells: evidence for two distinct Ca2+-dependent pathways in fission yeast. Mol Biol Cell. 2006 Nov;17(11):4790-800. Epub 2006 Aug 23.
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. |
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