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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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Cmk1 phosphorylates and binds Prz1. (A) Mobility shift of the Prz1 protein was analysed by Western blot using anti-GFP antibodies from Δcmk1 cells (Δcmk1) and wild-type cells untreated (wt) or treated with 100 mM of CaCl2 (wt+Ca2+) carrying the endogenous GFP-prz1 tagged gene (top). Anti-PSTAIR antibody was used to probe Cdc2 as a loading control (bottom). (B) Cmk1 and Cmk1-KA were overexpressed in GFP-Prz1 cells harbouring pREP1-Cmk1 or pREP1-Cmk1-KA plasmids in the absence of thiamine. The Prz1 protein was analysed by Western blot using anti-GFP antibodies (top). The level of Cmk1 and Cmk1-KA overexpression was detected using the anti-HA antibodies (bottom). (C) In vivo Prz1 phosphorylation by Cmk1. Prz1 was immunoprecipitated using anti-GFP antibodies from Δcmk1 and wild-type cells untreated (wt) or treated with 100 mM CaCl2 and 0.5 μg/ml of FK506 (top). Phosphorylation of Prz1 was detected with anti-Phospho Akt substrate (RXXS/T) (bottom). (D) Cmk1 protein was immunoprecipitated using anti-HA antibody from GFP-Prz1 cells harbouring the plasmid pREP1-Cmk1 or the empty plasmid pREP1 in the absence of thiamine (right top). Binding of Prz1 was detected using anti-GFP antibodies (right bottom). Left panels indicate the input levels of Prz1 (top), Cmk1 (middle) and Cdc2 as a loading control (bottom) detected with anti-GFP, anti-HA anti-PSTAIR antibodies, respectively.
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Figure 5: Cmk1 phosphorylates and binds Prz1. (A) Mobility shift of the Prz1 protein was analysed by Western blot using anti-GFP antibodies from Δcmk1 cells (Δcmk1) and wild-type cells untreated (wt) or treated with 100 mM of CaCl2 (wt+Ca2+) carrying the endogenous GFP-prz1 tagged gene (top). Anti-PSTAIR antibody was used to probe Cdc2 as a loading control (bottom). (B) Cmk1 and Cmk1-KA were overexpressed in GFP-Prz1 cells harbouring pREP1-Cmk1 or pREP1-Cmk1-KA plasmids in the absence of thiamine. The Prz1 protein was analysed by Western blot using anti-GFP antibodies (top). The level of Cmk1 and Cmk1-KA overexpression was detected using the anti-HA antibodies (bottom). (C) In vivo Prz1 phosphorylation by Cmk1. Prz1 was immunoprecipitated using anti-GFP antibodies from Δcmk1 and wild-type cells untreated (wt) or treated with 100 mM CaCl2 and 0.5 μg/ml of FK506 (top). Phosphorylation of Prz1 was detected with anti-Phospho Akt substrate (RXXS/T) (bottom). (D) Cmk1 protein was immunoprecipitated using anti-HA antibody from GFP-Prz1 cells harbouring the plasmid pREP1-Cmk1 or the empty plasmid pREP1 in the absence of thiamine (right top). Binding of Prz1 was detected using anti-GFP antibodies (right bottom). Left panels indicate the input levels of Prz1 (top), Cmk1 (middle) and Cdc2 as a loading control (bottom) detected with anti-GFP, anti-HA anti-PSTAIR antibodies, respectively.

Mentions: Prz1 activation and subcellular localisation are regulated by its phosphorylation state. In response to Ca2+ stimulation, Prz1 is dephosphorylated and activated (18). This activation can be monitored by a change in electrophoretic mobility, in addition to nuclear accumulation. We analysed the Prz1 protein in Δcmk1 cells and the mobility shift of Prz1 observed in wild-type cells is abolished in the absence of Cmk1 (Figure 5A). Moreover, we overexpressed Cmk1 and the inactive Cmk1-KA in wild-type cells. As Figure 5B shows, when Cmk1 was overexpressed, the Prz1 protein exhibited a slow electrophoretic-mobility which was absent with overexpression of Cmk1-KA, indicating that Prz1 is hyperphosphorylated when Cmk1 is overexpressed (Figure 5B).


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)

Cmk1 phosphorylates and binds Prz1. (A) Mobility shift of the Prz1 protein was analysed by Western blot using anti-GFP antibodies from Δcmk1 cells (Δcmk1) and wild-type cells untreated (wt) or treated with 100 mM of CaCl2 (wt+Ca2+) carrying the endogenous GFP-prz1 tagged gene (top). Anti-PSTAIR antibody was used to probe Cdc2 as a loading control (bottom). (B) Cmk1 and Cmk1-KA were overexpressed in GFP-Prz1 cells harbouring pREP1-Cmk1 or pREP1-Cmk1-KA plasmids in the absence of thiamine. The Prz1 protein was analysed by Western blot using anti-GFP antibodies (top). The level of Cmk1 and Cmk1-KA overexpression was detected using the anti-HA antibodies (bottom). (C) In vivo Prz1 phosphorylation by Cmk1. Prz1 was immunoprecipitated using anti-GFP antibodies from Δcmk1 and wild-type cells untreated (wt) or treated with 100 mM CaCl2 and 0.5 μg/ml of FK506 (top). Phosphorylation of Prz1 was detected with anti-Phospho Akt substrate (RXXS/T) (bottom). (D) Cmk1 protein was immunoprecipitated using anti-HA antibody from GFP-Prz1 cells harbouring the plasmid pREP1-Cmk1 or the empty plasmid pREP1 in the absence of thiamine (right top). Binding of Prz1 was detected using anti-GFP antibodies (right bottom). Left panels indicate the input levels of Prz1 (top), Cmk1 (middle) and Cdc2 as a loading control (bottom) detected with anti-GFP, anti-HA anti-PSTAIR antibodies, respectively.
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Related In: Results  -  Collection

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Figure 5: Cmk1 phosphorylates and binds Prz1. (A) Mobility shift of the Prz1 protein was analysed by Western blot using anti-GFP antibodies from Δcmk1 cells (Δcmk1) and wild-type cells untreated (wt) or treated with 100 mM of CaCl2 (wt+Ca2+) carrying the endogenous GFP-prz1 tagged gene (top). Anti-PSTAIR antibody was used to probe Cdc2 as a loading control (bottom). (B) Cmk1 and Cmk1-KA were overexpressed in GFP-Prz1 cells harbouring pREP1-Cmk1 or pREP1-Cmk1-KA plasmids in the absence of thiamine. The Prz1 protein was analysed by Western blot using anti-GFP antibodies (top). The level of Cmk1 and Cmk1-KA overexpression was detected using the anti-HA antibodies (bottom). (C) In vivo Prz1 phosphorylation by Cmk1. Prz1 was immunoprecipitated using anti-GFP antibodies from Δcmk1 and wild-type cells untreated (wt) or treated with 100 mM CaCl2 and 0.5 μg/ml of FK506 (top). Phosphorylation of Prz1 was detected with anti-Phospho Akt substrate (RXXS/T) (bottom). (D) Cmk1 protein was immunoprecipitated using anti-HA antibody from GFP-Prz1 cells harbouring the plasmid pREP1-Cmk1 or the empty plasmid pREP1 in the absence of thiamine (right top). Binding of Prz1 was detected using anti-GFP antibodies (right bottom). Left panels indicate the input levels of Prz1 (top), Cmk1 (middle) and Cdc2 as a loading control (bottom) detected with anti-GFP, anti-HA anti-PSTAIR antibodies, respectively.
Mentions: Prz1 activation and subcellular localisation are regulated by its phosphorylation state. In response to Ca2+ stimulation, Prz1 is dephosphorylated and activated (18). This activation can be monitored by a change in electrophoretic mobility, in addition to nuclear accumulation. We analysed the Prz1 protein in Δcmk1 cells and the mobility shift of Prz1 observed in wild-type cells is abolished in the absence of Cmk1 (Figure 5A). Moreover, we overexpressed Cmk1 and the inactive Cmk1-KA in wild-type cells. As Figure 5B shows, when Cmk1 was overexpressed, the Prz1 protein exhibited a slow electrophoretic-mobility which was absent with overexpression of Cmk1-KA, indicating that Prz1 is hyperphosphorylated when Cmk1 is overexpressed (Figure 5B).

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