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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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Overexpression of Cmk1 causes cell cycle arrest through Cdc25. (A) and (B) Wild-type (wt) and cdc25–9A cells containing the plasmid pREP1-cmk1-TD were grown either in the presence (+B1) or absence (-B1) of thiamine on (A) plates or (B) in liquid media. Bar, 10 μm. (C) Western blot of extracts from wild-type and cdc25–9A cells expressing pREP1-cmk1-TD grown either in the presence (+B1) or absence (-B1) of thiamine were probed with anti-Cdc25 antibodies (top), anti-HA antibodies to detect Cmk1-TD (middle) and anti-PSTAIR antibodies to detect Cdc2 as a loading control. * indicates unspecific bands. (D) Time course of wild-type (wt) and Δcmk1 cells exposed to 100 mM CaCl2. The Cdc25 protein level was analysed at the times indicated by Western blot using anti-Cdc25 antibodies (top) and Cdc2 was probed as a loading control with anti-PSTAIR antibodies (bottom).
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Figure 7: Overexpression of Cmk1 causes cell cycle arrest through Cdc25. (A) and (B) Wild-type (wt) and cdc25–9A cells containing the plasmid pREP1-cmk1-TD were grown either in the presence (+B1) or absence (-B1) of thiamine on (A) plates or (B) in liquid media. Bar, 10 μm. (C) Western blot of extracts from wild-type and cdc25–9A cells expressing pREP1-cmk1-TD grown either in the presence (+B1) or absence (-B1) of thiamine were probed with anti-Cdc25 antibodies (top), anti-HA antibodies to detect Cmk1-TD (middle) and anti-PSTAIR antibodies to detect Cdc2 as a loading control. * indicates unspecific bands. (D) Time course of wild-type (wt) and Δcmk1 cells exposed to 100 mM CaCl2. The Cdc25 protein level was analysed at the times indicated by Western blot using anti-Cdc25 antibodies (top) and Cdc2 was probed as a loading control with anti-PSTAIR antibodies (bottom).

Mentions: Expression of the constitutively active form of Cmk1 (Cmk1-T192D) arrests cell cycle progression (Figure 7A) (9). We and others have previously reported that activation of members of the CaM-dependent kinase family, such as Chk1, Cds1 or Srk1, causes cell cycle arrest dependent on Cdc25 phosphorylation (37–41). We therefore studied whether the cell cycle arrest caused by the overexpression of Cmk1-T192D is dependent on Cdc25 phosphorylation. To this end, we overexpressed Cmk1-T192D in a strain carrying the cdc25–9A allele, which contains mutations at 9 of the consensus sites of CaM-dependent kinases (40,42–43). The growth arrest and cell elongation typical of cell cycle arrest caused by the overexpression of Cmk1-T192D were suppressed when Cmk1-T192D was overexpressed in cdc2–59A cells (Figure 7A and B).


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)

Overexpression of Cmk1 causes cell cycle arrest through Cdc25. (A) and (B) Wild-type (wt) and cdc25–9A cells containing the plasmid pREP1-cmk1-TD were grown either in the presence (+B1) or absence (-B1) of thiamine on (A) plates or (B) in liquid media. Bar, 10 μm. (C) Western blot of extracts from wild-type and cdc25–9A cells expressing pREP1-cmk1-TD grown either in the presence (+B1) or absence (-B1) of thiamine were probed with anti-Cdc25 antibodies (top), anti-HA antibodies to detect Cmk1-TD (middle) and anti-PSTAIR antibodies to detect Cdc2 as a loading control. * indicates unspecific bands. (D) Time course of wild-type (wt) and Δcmk1 cells exposed to 100 mM CaCl2. The Cdc25 protein level was analysed at the times indicated by Western blot using anti-Cdc25 antibodies (top) and Cdc2 was probed as a loading control with anti-PSTAIR antibodies (bottom).
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Related In: Results  -  Collection

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Figure 7: Overexpression of Cmk1 causes cell cycle arrest through Cdc25. (A) and (B) Wild-type (wt) and cdc25–9A cells containing the plasmid pREP1-cmk1-TD were grown either in the presence (+B1) or absence (-B1) of thiamine on (A) plates or (B) in liquid media. Bar, 10 μm. (C) Western blot of extracts from wild-type and cdc25–9A cells expressing pREP1-cmk1-TD grown either in the presence (+B1) or absence (-B1) of thiamine were probed with anti-Cdc25 antibodies (top), anti-HA antibodies to detect Cmk1-TD (middle) and anti-PSTAIR antibodies to detect Cdc2 as a loading control. * indicates unspecific bands. (D) Time course of wild-type (wt) and Δcmk1 cells exposed to 100 mM CaCl2. The Cdc25 protein level was analysed at the times indicated by Western blot using anti-Cdc25 antibodies (top) and Cdc2 was probed as a loading control with anti-PSTAIR antibodies (bottom).
Mentions: Expression of the constitutively active form of Cmk1 (Cmk1-T192D) arrests cell cycle progression (Figure 7A) (9). We and others have previously reported that activation of members of the CaM-dependent kinase family, such as Chk1, Cds1 or Srk1, causes cell cycle arrest dependent on Cdc25 phosphorylation (37–41). We therefore studied whether the cell cycle arrest caused by the overexpression of Cmk1-T192D is dependent on Cdc25 phosphorylation. To this end, we overexpressed Cmk1-T192D in a strain carrying the cdc25–9A allele, which contains mutations at 9 of the consensus sites of CaM-dependent kinases (40,42–43). The growth arrest and cell elongation typical of cell cycle arrest caused by the overexpression of Cmk1-T192D were suppressed when Cmk1-T192D was overexpressed in cdc2–59A cells (Figure 7A and B).

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