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Transient receptor potential (TRP) and Cch1-Yam8 channels play key roles in the regulation of cytoplasmic Ca2+ in fission yeast.

Ma Y, Sugiura R, Koike A, Ebina H, Sio SO, Kuno T - PLoS ONE (2011)

Bottom Line: We found that the extracellularly added Ca(2+) caused a dose-dependent increase in the cytoplasmic Ca(2+) level and resulted in a burst-like peak.We also found that extracellularly added NaCl plus FK506 caused a synergistic cytosolic Ca(2+) increase that is dependent on the inhibition of calcineurin activity, but not on the inhibition of Prz1.These results suggest that Pmk1 MAPK and calcineurin positively and negatively regulate the Cch1-Yam8 complex, respectively, via modulating the balance between phosphorylation and dyphosphorylation state.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pharmacology and Pharmacogenomics, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Japan. mayan@med.kobe-u.ac.jp

ABSTRACT
The regulation of cytoplasmic Ca(2+) is crucial for various cellular processes. Here, we examined the cytoplasmic Ca(2+) levels in living fission yeast cells by a highly sensitive bioluminescence resonance energy transfer-based assay using GFP-aequorin fusion protein linked by 19 amino acid. We monitored the cytoplasmic Ca(2+) level and its change caused by extracellular stimulants such as CaCl(2) or NaCl plus FK506 (calcineurin inhibitor). We found that the extracellularly added Ca(2+) caused a dose-dependent increase in the cytoplasmic Ca(2+) level and resulted in a burst-like peak. The overexpression of two transient receptor potential (TRP) channel homologues, Trp1322 or Pkd2, markedly enhanced this response. Interestingly, the burst-like peak upon TRP overexpression was completely abolished by gene deletion of calcineurin and was dramatically decreased by gene deletion of Prz1, a downstream transcription factor activated by calcineurin. Furthermore, 1 hour treatment with FK506 failed to suppress the burst-like peak. These results suggest that the burst-like Ca(2+) peak is dependent on the transcriptional activity of Prz1, but not on the direct TRP dephosphorylation. We also found that extracellularly added NaCl plus FK506 caused a synergistic cytosolic Ca(2+) increase that is dependent on the inhibition of calcineurin activity, but not on the inhibition of Prz1. The synergistic Ca(2+) increase is abolished by the addition of the Ca(2+) chelator BAPTA into the media, and is also abolished by deletion of the gene encoding a subunit of the Cch1-Yam8 Ca(2+) channel complex, indicating that the synergistic increase is caused by the Ca(2+) influx from the extracellular medium via the Cch1-Yam8 complex. Furthermore, deletion of Pmk1 MAPK abolished the Ca(2+) influx, and overexpression of the constitutively active Pek1 MAPKK enhanced the influx. These results suggest that Pmk1 MAPK and calcineurin positively and negatively regulate the Cch1-Yam8 complex, respectively, via modulating the balance between phosphorylation and dyphosphorylation state.

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A cartoon figure illustrating a molecular mechanism of Ca2+ signaling.The cell wall damaging agents (e.g. NaCl, KCl, MgCl2) activate the cell wall integrity MAPK Pmk1, then the activated Pmk1 phosphorylates Cch1, resulting in the opening of the Cch1-Yam8 channel and in Ca2+ influx. In the absence of FK506, the increase in the cytoplasmic Ca2+ level activates calcineurin, which in turn dephosphorylates Cch1, resulting in the closing of the channel. In the presence of FK506, calcineurin is inhibited, thus resulting in the persistent opening of the Cch1-Yam8 channels. Trp1322 and Pkd2 directly regulates calcium influx, and also may crosstalk with other components of the Pmk1 MAPK pathway upstream of Pek1, thus indirectly affecting the Cch1-Yam8 complex upon NaCl plus FK506 stimulation.
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pone-0022421-g008: A cartoon figure illustrating a molecular mechanism of Ca2+ signaling.The cell wall damaging agents (e.g. NaCl, KCl, MgCl2) activate the cell wall integrity MAPK Pmk1, then the activated Pmk1 phosphorylates Cch1, resulting in the opening of the Cch1-Yam8 channel and in Ca2+ influx. In the absence of FK506, the increase in the cytoplasmic Ca2+ level activates calcineurin, which in turn dephosphorylates Cch1, resulting in the closing of the channel. In the presence of FK506, calcineurin is inhibited, thus resulting in the persistent opening of the Cch1-Yam8 channels. Trp1322 and Pkd2 directly regulates calcium influx, and also may crosstalk with other components of the Pmk1 MAPK pathway upstream of Pek1, thus indirectly affecting the Cch1-Yam8 complex upon NaCl plus FK506 stimulation.

Mentions: It has been reported that in budding yeast calcineurin can directly dephosphorylate Cch1, resulting in the inhibition of the Cch1-Mid1 channel [29]. In the present study, we summarize our hypothesis in Figure 8. As shown in the figure, the NaCl addition damages the cell wall and activates the cell wall integrity MAPK Pmk1, and the activated Pmk1 phosphorylates Cch1, resulting in the opening of the Cch1-Yam8 channel and leading to a Ca2+ influx. In the absence of FK506, the cytosolic Ca2+ increase activates calcineurin, which in turn dephosphorylates Cch1, resulting in the closing of the channel. In the presence of FK506, FK506 inhibits calcineurin, thus resulting in the persistent opening of the Cch1-Yam8 channels.


Transient receptor potential (TRP) and Cch1-Yam8 channels play key roles in the regulation of cytoplasmic Ca2+ in fission yeast.

Ma Y, Sugiura R, Koike A, Ebina H, Sio SO, Kuno T - PLoS ONE (2011)

A cartoon figure illustrating a molecular mechanism of Ca2+ signaling.The cell wall damaging agents (e.g. NaCl, KCl, MgCl2) activate the cell wall integrity MAPK Pmk1, then the activated Pmk1 phosphorylates Cch1, resulting in the opening of the Cch1-Yam8 channel and in Ca2+ influx. In the absence of FK506, the increase in the cytoplasmic Ca2+ level activates calcineurin, which in turn dephosphorylates Cch1, resulting in the closing of the channel. In the presence of FK506, calcineurin is inhibited, thus resulting in the persistent opening of the Cch1-Yam8 channels. Trp1322 and Pkd2 directly regulates calcium influx, and also may crosstalk with other components of the Pmk1 MAPK pathway upstream of Pek1, thus indirectly affecting the Cch1-Yam8 complex upon NaCl plus FK506 stimulation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139647&req=5

pone-0022421-g008: A cartoon figure illustrating a molecular mechanism of Ca2+ signaling.The cell wall damaging agents (e.g. NaCl, KCl, MgCl2) activate the cell wall integrity MAPK Pmk1, then the activated Pmk1 phosphorylates Cch1, resulting in the opening of the Cch1-Yam8 channel and in Ca2+ influx. In the absence of FK506, the increase in the cytoplasmic Ca2+ level activates calcineurin, which in turn dephosphorylates Cch1, resulting in the closing of the channel. In the presence of FK506, calcineurin is inhibited, thus resulting in the persistent opening of the Cch1-Yam8 channels. Trp1322 and Pkd2 directly regulates calcium influx, and also may crosstalk with other components of the Pmk1 MAPK pathway upstream of Pek1, thus indirectly affecting the Cch1-Yam8 complex upon NaCl plus FK506 stimulation.
Mentions: It has been reported that in budding yeast calcineurin can directly dephosphorylate Cch1, resulting in the inhibition of the Cch1-Mid1 channel [29]. In the present study, we summarize our hypothesis in Figure 8. As shown in the figure, the NaCl addition damages the cell wall and activates the cell wall integrity MAPK Pmk1, and the activated Pmk1 phosphorylates Cch1, resulting in the opening of the Cch1-Yam8 channel and leading to a Ca2+ influx. In the absence of FK506, the cytosolic Ca2+ increase activates calcineurin, which in turn dephosphorylates Cch1, resulting in the closing of the channel. In the presence of FK506, FK506 inhibits calcineurin, thus resulting in the persistent opening of the Cch1-Yam8 channels.

Bottom Line: We found that the extracellularly added Ca(2+) caused a dose-dependent increase in the cytoplasmic Ca(2+) level and resulted in a burst-like peak.We also found that extracellularly added NaCl plus FK506 caused a synergistic cytosolic Ca(2+) increase that is dependent on the inhibition of calcineurin activity, but not on the inhibition of Prz1.These results suggest that Pmk1 MAPK and calcineurin positively and negatively regulate the Cch1-Yam8 complex, respectively, via modulating the balance between phosphorylation and dyphosphorylation state.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pharmacology and Pharmacogenomics, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Japan. mayan@med.kobe-u.ac.jp

ABSTRACT
The regulation of cytoplasmic Ca(2+) is crucial for various cellular processes. Here, we examined the cytoplasmic Ca(2+) levels in living fission yeast cells by a highly sensitive bioluminescence resonance energy transfer-based assay using GFP-aequorin fusion protein linked by 19 amino acid. We monitored the cytoplasmic Ca(2+) level and its change caused by extracellular stimulants such as CaCl(2) or NaCl plus FK506 (calcineurin inhibitor). We found that the extracellularly added Ca(2+) caused a dose-dependent increase in the cytoplasmic Ca(2+) level and resulted in a burst-like peak. The overexpression of two transient receptor potential (TRP) channel homologues, Trp1322 or Pkd2, markedly enhanced this response. Interestingly, the burst-like peak upon TRP overexpression was completely abolished by gene deletion of calcineurin and was dramatically decreased by gene deletion of Prz1, a downstream transcription factor activated by calcineurin. Furthermore, 1 hour treatment with FK506 failed to suppress the burst-like peak. These results suggest that the burst-like Ca(2+) peak is dependent on the transcriptional activity of Prz1, but not on the direct TRP dephosphorylation. We also found that extracellularly added NaCl plus FK506 caused a synergistic cytosolic Ca(2+) increase that is dependent on the inhibition of calcineurin activity, but not on the inhibition of Prz1. The synergistic Ca(2+) increase is abolished by the addition of the Ca(2+) chelator BAPTA into the media, and is also abolished by deletion of the gene encoding a subunit of the Cch1-Yam8 Ca(2+) channel complex, indicating that the synergistic increase is caused by the Ca(2+) influx from the extracellular medium via the Cch1-Yam8 complex. Furthermore, deletion of Pmk1 MAPK abolished the Ca(2+) influx, and overexpression of the constitutively active Pek1 MAPKK enhanced the influx. These results suggest that Pmk1 MAPK and calcineurin positively and negatively regulate the Cch1-Yam8 complex, respectively, via modulating the balance between phosphorylation and dyphosphorylation state.

Show MeSH
Related in: MedlinePlus