Limits...
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.

Show MeSH

Related in: MedlinePlus

Cmk1 regulates Prz1 transcriptional activity. (A) Wild-type and Δcmk1 strains transformed with the CDRE reporter vector were incubated with d-luciferun and untreated (no stimuli) and treated with either 20 mM or 200 mM CaCl2. Using a luminometer, light emission levels expressed as relative light units were measured up to 120 min. The graph ‘no stimuli’ represents the values of cells starting exponential growth. The data shown represent multiple experiments. (B) Wild-type cells harbouring the integration plasmid (CDRE-TATAbox-luc1) (KP2513 strain) were transformed with the multicopy plasmid empty (vector) or carrying the cmk1 gene (Cmk1 OP). Cells were incubated with d-luciferin and treated with various concentrations of CaCl2 as indicated. As in (A) light emission levels expressed as relative light units were measured up to 120 min. The graph ‘no stimuli’ represents the values of cells starting exponential growth. The data shown represent multiple experiments. The graphics on the right in A and B represent the overall peak heights of the relative light units measured from the graphs on the left. (C) Cells expressing chromosomal GFP-Prz1 under the control of the nmt promoter were treated (+) or untreated (-) with 100 mM CaCl2 and Cmk1 (pREP1-cmk1) or the inactive Cmk1-KA (pREP1-cmk1-KA) were overexpressed under the control of nmt promoter by growing the cells in EMM without thiamine (-B1). The graph represents the quantification of GFP-Prz1 localisation in each indicated condition of three different experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150787&req=5

Figure 4: Cmk1 regulates Prz1 transcriptional activity. (A) Wild-type and Δcmk1 strains transformed with the CDRE reporter vector were incubated with d-luciferun and untreated (no stimuli) and treated with either 20 mM or 200 mM CaCl2. Using a luminometer, light emission levels expressed as relative light units were measured up to 120 min. The graph ‘no stimuli’ represents the values of cells starting exponential growth. The data shown represent multiple experiments. (B) Wild-type cells harbouring the integration plasmid (CDRE-TATAbox-luc1) (KP2513 strain) were transformed with the multicopy plasmid empty (vector) or carrying the cmk1 gene (Cmk1 OP). Cells were incubated with d-luciferin and treated with various concentrations of CaCl2 as indicated. As in (A) light emission levels expressed as relative light units were measured up to 120 min. The graph ‘no stimuli’ represents the values of cells starting exponential growth. The data shown represent multiple experiments. The graphics on the right in A and B represent the overall peak heights of the relative light units measured from the graphs on the left. (C) Cells expressing chromosomal GFP-Prz1 under the control of the nmt promoter were treated (+) or untreated (-) with 100 mM CaCl2 and Cmk1 (pREP1-cmk1) or the inactive Cmk1-KA (pREP1-cmk1-KA) were overexpressed under the control of nmt promoter by growing the cells in EMM without thiamine (-B1). The graph represents the quantification of GFP-Prz1 localisation in each indicated condition of three different experiments.

Mentions: In budding yeast and mammalian cells, the activity of Crz1 and NFAT transcription factors, is regulated by different signalling pathways which affect kinases (14). This prompted us to examine whether Cmk1 is involved in negative regulation of Prz1 by analysing the Ca2+-induced CDRE transcriptional activity of Prz1 in Δcmk1 cells compared to wild-type. The reporter response of Δcmk1 cells was markedly enhanced, reflecting the increase of Prz1 transcriptional activity compared with wild-type cells (Figure 4A). In contrast, overexpression of Cmk1 suppressed the Prz1 transcriptional activity (Figure 4B), indicating that Cmk1 is indeed involved in the negative regulation of Prz1.


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 regulates Prz1 transcriptional activity. (A) Wild-type and Δcmk1 strains transformed with the CDRE reporter vector were incubated with d-luciferun and untreated (no stimuli) and treated with either 20 mM or 200 mM CaCl2. Using a luminometer, light emission levels expressed as relative light units were measured up to 120 min. The graph ‘no stimuli’ represents the values of cells starting exponential growth. The data shown represent multiple experiments. (B) Wild-type cells harbouring the integration plasmid (CDRE-TATAbox-luc1) (KP2513 strain) were transformed with the multicopy plasmid empty (vector) or carrying the cmk1 gene (Cmk1 OP). Cells were incubated with d-luciferin and treated with various concentrations of CaCl2 as indicated. As in (A) light emission levels expressed as relative light units were measured up to 120 min. The graph ‘no stimuli’ represents the values of cells starting exponential growth. The data shown represent multiple experiments. The graphics on the right in A and B represent the overall peak heights of the relative light units measured from the graphs on the left. (C) Cells expressing chromosomal GFP-Prz1 under the control of the nmt promoter were treated (+) or untreated (-) with 100 mM CaCl2 and Cmk1 (pREP1-cmk1) or the inactive Cmk1-KA (pREP1-cmk1-KA) were overexpressed under the control of nmt promoter by growing the cells in EMM without thiamine (-B1). The graph represents the quantification of GFP-Prz1 localisation in each indicated condition of three different experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Cmk1 regulates Prz1 transcriptional activity. (A) Wild-type and Δcmk1 strains transformed with the CDRE reporter vector were incubated with d-luciferun and untreated (no stimuli) and treated with either 20 mM or 200 mM CaCl2. Using a luminometer, light emission levels expressed as relative light units were measured up to 120 min. The graph ‘no stimuli’ represents the values of cells starting exponential growth. The data shown represent multiple experiments. (B) Wild-type cells harbouring the integration plasmid (CDRE-TATAbox-luc1) (KP2513 strain) were transformed with the multicopy plasmid empty (vector) or carrying the cmk1 gene (Cmk1 OP). Cells were incubated with d-luciferin and treated with various concentrations of CaCl2 as indicated. As in (A) light emission levels expressed as relative light units were measured up to 120 min. The graph ‘no stimuli’ represents the values of cells starting exponential growth. The data shown represent multiple experiments. The graphics on the right in A and B represent the overall peak heights of the relative light units measured from the graphs on the left. (C) Cells expressing chromosomal GFP-Prz1 under the control of the nmt promoter were treated (+) or untreated (-) with 100 mM CaCl2 and Cmk1 (pREP1-cmk1) or the inactive Cmk1-KA (pREP1-cmk1-KA) were overexpressed under the control of nmt promoter by growing the cells in EMM without thiamine (-B1). The graph represents the quantification of GFP-Prz1 localisation in each indicated condition of three different experiments.
Mentions: In budding yeast and mammalian cells, the activity of Crz1 and NFAT transcription factors, is regulated by different signalling pathways which affect kinases (14). This prompted us to examine whether Cmk1 is involved in negative regulation of Prz1 by analysing the Ca2+-induced CDRE transcriptional activity of Prz1 in Δcmk1 cells compared to wild-type. The reporter response of Δcmk1 cells was markedly enhanced, reflecting the increase of Prz1 transcriptional activity compared with wild-type cells (Figure 4A). In contrast, overexpression of Cmk1 suppressed the Prz1 transcriptional activity (Figure 4B), indicating that Cmk1 is indeed involved in the negative regulation of Prz1.

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