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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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Suppression of Ca2+ resistance of cmk1 deleted cells by elimination of calcineurin pathway. (A) Ca2+ sensitivity. Wild-type and Δcmk1 cells were grown in YES medium and spotted on YES plates containing different concentrations of CaCl2 and incubated for 3 days at 30°C. (B) Δcmk1 cells containing the multicopy plasmid pREP1, empty (pREP1) or carrying different versions of cmk1 gene (pREP1-cmk1, pREP1-cmk1-T192D and pREP1-cmk1-T192D-K60A) were spotted onto EMM plates with thiamine (+B1), without thiamine (-B1) and without thiamine plus CaCl2 (-B1+ 100mM CaCl2) and were incubated for 4 day at 30°C. (C) and (D) The indicated strains were grown in YES medium and spotted on YES plates containing different concentrations of CaCl2 and incubated for 3 days at 30°C. Lower CaCl2 concentrations of figures C and D are shown in Supplementary Figure S1.
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Figure 6: Suppression of Ca2+ resistance of cmk1 deleted cells by elimination of calcineurin pathway. (A) Ca2+ sensitivity. Wild-type and Δcmk1 cells were grown in YES medium and spotted on YES plates containing different concentrations of CaCl2 and incubated for 3 days at 30°C. (B) Δcmk1 cells containing the multicopy plasmid pREP1, empty (pREP1) or carrying different versions of cmk1 gene (pREP1-cmk1, pREP1-cmk1-T192D and pREP1-cmk1-T192D-K60A) were spotted onto EMM plates with thiamine (+B1), without thiamine (-B1) and without thiamine plus CaCl2 (-B1+ 100mM CaCl2) and were incubated for 4 day at 30°C. (C) and (D) The indicated strains were grown in YES medium and spotted on YES plates containing different concentrations of CaCl2 and incubated for 3 days at 30°C. Lower CaCl2 concentrations of figures C and D are shown in Supplementary Figure S1.

Mentions: The above results imply a role of Cmk1 in the control of Ca2+ homeostasis. Therefore, we screened the growth of wild-type and Δcmk1 cells at different concentrations of Ca2+. As shown in Figure 6A, Δcmk1 cells displayed a resistant phenotype to high Ca2+ concentrations. This observation is consistent with its role as a negative regulator of Prz1 (Figure 6A). In contrast, overexpression of the constitutively active form of Cmk1 (Cmk1-T192D) was capable of suppressing Ca2+ resistance (Figure 6B). The sensitivity to Ca2+ acquired with Cmk1-T192D expression was associated with Cmk1 activity because overexpression of the catalytically inactive Cmk1-T192D, which has the lysine 60 from the ATP-binding domain mutated to alanine (Cmk1-T192D-K60A), rendered it insensitive to Ca2+ (Figure 6B).


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)

Suppression of Ca2+ resistance of cmk1 deleted cells by elimination of calcineurin pathway. (A) Ca2+ sensitivity. Wild-type and Δcmk1 cells were grown in YES medium and spotted on YES plates containing different concentrations of CaCl2 and incubated for 3 days at 30°C. (B) Δcmk1 cells containing the multicopy plasmid pREP1, empty (pREP1) or carrying different versions of cmk1 gene (pREP1-cmk1, pREP1-cmk1-T192D and pREP1-cmk1-T192D-K60A) were spotted onto EMM plates with thiamine (+B1), without thiamine (-B1) and without thiamine plus CaCl2 (-B1+ 100mM CaCl2) and were incubated for 4 day at 30°C. (C) and (D) The indicated strains were grown in YES medium and spotted on YES plates containing different concentrations of CaCl2 and incubated for 3 days at 30°C. Lower CaCl2 concentrations of figures C and D are shown in Supplementary Figure S1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Suppression of Ca2+ resistance of cmk1 deleted cells by elimination of calcineurin pathway. (A) Ca2+ sensitivity. Wild-type and Δcmk1 cells were grown in YES medium and spotted on YES plates containing different concentrations of CaCl2 and incubated for 3 days at 30°C. (B) Δcmk1 cells containing the multicopy plasmid pREP1, empty (pREP1) or carrying different versions of cmk1 gene (pREP1-cmk1, pREP1-cmk1-T192D and pREP1-cmk1-T192D-K60A) were spotted onto EMM plates with thiamine (+B1), without thiamine (-B1) and without thiamine plus CaCl2 (-B1+ 100mM CaCl2) and were incubated for 4 day at 30°C. (C) and (D) The indicated strains were grown in YES medium and spotted on YES plates containing different concentrations of CaCl2 and incubated for 3 days at 30°C. Lower CaCl2 concentrations of figures C and D are shown in Supplementary Figure S1.
Mentions: The above results imply a role of Cmk1 in the control of Ca2+ homeostasis. Therefore, we screened the growth of wild-type and Δcmk1 cells at different concentrations of Ca2+. As shown in Figure 6A, Δcmk1 cells displayed a resistant phenotype to high Ca2+ concentrations. This observation is consistent with its role as a negative regulator of Prz1 (Figure 6A). In contrast, overexpression of the constitutively active form of Cmk1 (Cmk1-T192D) was capable of suppressing Ca2+ resistance (Figure 6B). The sensitivity to Ca2+ acquired with Cmk1-T192D expression was associated with Cmk1 activity because overexpression of the catalytically inactive Cmk1-T192D, which has the lysine 60 from the ATP-binding domain mutated to alanine (Cmk1-T192D-K60A), rendered it insensitive to Ca2+ (Figure 6B).

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