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.
Deng
Lu
L
Division of Molecular Pharmacology and Pharmacogenomics, Department of Genome Sciences, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
Sugiura
Reiko
R
Takeuchi
Mai
M
Suzuki
Masahiro
M
Ebina
Hidemine
H
Takami
Tomonori
T
Koike
Atsushi
A
Iba
Shiori
S
Kuno
Takayoshi
T
D019703:Calcineurin / Q000235:genetics / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000378:metabolism; D002746:Chlorpromazine / Q000494:pharmacology; D054714:Echinocandins; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D015870:Gene Expression; D005800:Genes, Fungal / Q000235:genetics; D006207:Half-Life; D055666:Lipopeptides; D008074:Lipoproteins / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D000077551:Micafungin; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010456:Peptides, Cyclic / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D012568:Schizosaccharomyces / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology
D018797:Cell Cycle Proteins; D054714:Echinocandins; D055666:Lipopeptides; D008074:Lipoproteins; D008562:Membrane Glycoproteins; D010456:Peptides, Cyclic; D029702:Schizosaccharomyces pombe Proteins; C405644:YAM8 protein, S pombe; D012965:Sodium Chloride; D008156:Luciferases; C119549:Mek1 protein, S pombe; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D019703:Calcineurin; D002122:Calcium Chloride; D000077551:Micafungin; D002746:Chlorpromazine