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Negative feedback regulation of calcineurin-dependent Prz1 transcription factor by the CaMKK-CaMK1 axis in fission yeast.

Cisneros-Barroso E, Yance-Chávez T, Kito A, Sugiura R, Gómez-Hierro A, Giménez-Zaragoza D, Aligue R - Nucleic Acids Res. (2014)

Bottom Line: Then, active Cmk1 binds, phosphorylates and inactivates Prz1 transcription activity whilst at the same time cmk1 expression is enhanced by Prz1 in response to Ca(2+).This work reveals that Cmk1 kinase activated by the newly identified Ckk2 counteracts calcineurin function by negatively regulating Prz1 activity which in turn is involved in activating cmk1 gene transcription.These results are the first insights into Cmk1 and Ckk2 function in Schizosaccharomyces pombe.

View Article: PubMed Central - PubMed

Affiliation: Departament de Biologia Cellular, Immunologia i Neurociències, Facultat de Medicina, Universitat de Barcelona, Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), Barcelona 08036, Catalunya, Spain.

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Increase of Cmk1 expression and phosphorylation due to Ca2+. (A) Time course of cells harbouring endogenous cmk1:HA gene exposed to 100 mM CaCl2. Cmk1 protein level was analysed at the times indicated by Western blot using anti-HA antibodies (top) and Cdc2 was probed as a loading control with anti-PSTAIR antibodies (A and B, bottom). (B) cmk1:HA cells exposed to 100 mM CaCl2 for 30 min were treated (+) or untreated (-) with EGTA before analysing the Cmk1 protein by Western blot using anti-HA antibodies (top). (C) Cell extracts of cells expressing Cmk1-HA in the presence of 100 mM CaCl2 were treated before electrophoresis with λ-protein phosphatase and with phosphatase inhibitors when indicated. Cmk1 was analysed by Western blot using anti-HA antibodies. (D) Cmk1 gene expression. The mRNA of cells treated (wt CaCl2) or untreated (wt) with 100 mM CaCl2 was extracted and the cmk1 mRNA level was analysed by RT-PCR using the oligonucleotides Cmk1 RT Fw and Rv.
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Figure 1: Increase of Cmk1 expression and phosphorylation due to Ca2+. (A) Time course of cells harbouring endogenous cmk1:HA gene exposed to 100 mM CaCl2. Cmk1 protein level was analysed at the times indicated by Western blot using anti-HA antibodies (top) and Cdc2 was probed as a loading control with anti-PSTAIR antibodies (A and B, bottom). (B) cmk1:HA cells exposed to 100 mM CaCl2 for 30 min were treated (+) or untreated (-) with EGTA before analysing the Cmk1 protein by Western blot using anti-HA antibodies (top). (C) Cell extracts of cells expressing Cmk1-HA in the presence of 100 mM CaCl2 were treated before electrophoresis with λ-protein phosphatase and with phosphatase inhibitors when indicated. Cmk1 was analysed by Western blot using anti-HA antibodies. (D) Cmk1 gene expression. The mRNA of cells treated (wt CaCl2) or untreated (wt) with 100 mM CaCl2 was extracted and the cmk1 mRNA level was analysed by RT-PCR using the oligonucleotides Cmk1 RT Fw and Rv.

Mentions: The Cmk1 protein was analysed in response to Ca2+. We found that Cmk1 protein levels increased during exposure to Ca2+ (Figure 1A). In addition, we observed the appearance of a slow migration band concomitant with exposure to Ca2+ (Figure 1A). To confirm the Ca2+-dependence of the slow Cmk1 migration band, growing cells were treated with Ca2+ and divided into two cultures, with Ca2+-chelator EGTA added to one of them. As Figure 1B shows, the slow migrating band was eliminated when EGTA was added to Ca2+-treated cells. To determine whether phosphorylation contributes to the Cmk1 slow migration band, we performed a phosphatase assay. Cmk1-HA was immunoprecipitated from Ca2+-treated extracts and incubated with or without λ-protein phosphatase. The Cmk1 mobility shift was then analysed. As shown in Figure 1C, phosphatase treatment greatly reduced the presence of the slow migration band, indicating that the Cmk1 protein is phosphorylated in response to Ca2+.


Negative feedback regulation of calcineurin-dependent Prz1 transcription factor by the CaMKK-CaMK1 axis in fission yeast.

Cisneros-Barroso E, Yance-Chávez T, Kito A, Sugiura R, Gómez-Hierro A, Giménez-Zaragoza D, Aligue R - Nucleic Acids Res. (2014)

Increase of Cmk1 expression and phosphorylation due to Ca2+. (A) Time course of cells harbouring endogenous cmk1:HA gene exposed to 100 mM CaCl2. Cmk1 protein level was analysed at the times indicated by Western blot using anti-HA antibodies (top) and Cdc2 was probed as a loading control with anti-PSTAIR antibodies (A and B, bottom). (B) cmk1:HA cells exposed to 100 mM CaCl2 for 30 min were treated (+) or untreated (-) with EGTA before analysing the Cmk1 protein by Western blot using anti-HA antibodies (top). (C) Cell extracts of cells expressing Cmk1-HA in the presence of 100 mM CaCl2 were treated before electrophoresis with λ-protein phosphatase and with phosphatase inhibitors when indicated. Cmk1 was analysed by Western blot using anti-HA antibodies. (D) Cmk1 gene expression. The mRNA of cells treated (wt CaCl2) or untreated (wt) with 100 mM CaCl2 was extracted and the cmk1 mRNA level was analysed by RT-PCR using the oligonucleotides Cmk1 RT Fw and Rv.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4150787&req=5

Figure 1: Increase of Cmk1 expression and phosphorylation due to Ca2+. (A) Time course of cells harbouring endogenous cmk1:HA gene exposed to 100 mM CaCl2. Cmk1 protein level was analysed at the times indicated by Western blot using anti-HA antibodies (top) and Cdc2 was probed as a loading control with anti-PSTAIR antibodies (A and B, bottom). (B) cmk1:HA cells exposed to 100 mM CaCl2 for 30 min were treated (+) or untreated (-) with EGTA before analysing the Cmk1 protein by Western blot using anti-HA antibodies (top). (C) Cell extracts of cells expressing Cmk1-HA in the presence of 100 mM CaCl2 were treated before electrophoresis with λ-protein phosphatase and with phosphatase inhibitors when indicated. Cmk1 was analysed by Western blot using anti-HA antibodies. (D) Cmk1 gene expression. The mRNA of cells treated (wt CaCl2) or untreated (wt) with 100 mM CaCl2 was extracted and the cmk1 mRNA level was analysed by RT-PCR using the oligonucleotides Cmk1 RT Fw and Rv.
Mentions: The Cmk1 protein was analysed in response to Ca2+. We found that Cmk1 protein levels increased during exposure to Ca2+ (Figure 1A). In addition, we observed the appearance of a slow migration band concomitant with exposure to Ca2+ (Figure 1A). To confirm the Ca2+-dependence of the slow Cmk1 migration band, growing cells were treated with Ca2+ and divided into two cultures, with Ca2+-chelator EGTA added to one of them. As Figure 1B shows, the slow migrating band was eliminated when EGTA was added to Ca2+-treated cells. To determine whether phosphorylation contributes to the Cmk1 slow migration band, we performed a phosphatase assay. Cmk1-HA was immunoprecipitated from Ca2+-treated extracts and incubated with or without λ-protein phosphatase. The Cmk1 mobility shift was then analysed. As shown in Figure 1C, phosphatase treatment greatly reduced the presence of the slow migration band, indicating that the Cmk1 protein is phosphorylated in response to Ca2+.

Bottom Line: Then, active Cmk1 binds, phosphorylates and inactivates Prz1 transcription activity whilst at the same time cmk1 expression is enhanced by Prz1 in response to Ca(2+).This work reveals that Cmk1 kinase activated by the newly identified Ckk2 counteracts calcineurin function by negatively regulating Prz1 activity which in turn is involved in activating cmk1 gene transcription.These results are the first insights into Cmk1 and Ckk2 function in Schizosaccharomyces pombe.

View Article: PubMed Central - PubMed

Affiliation: Departament de Biologia Cellular, Immunologia i Neurociències, Facultat de Medicina, Universitat de Barcelona, Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), Barcelona 08036, Catalunya, Spain.

Show MeSH
Related in: MedlinePlus