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Neuronal calcium sensor proteins are unable to modulate NFAT activation in mammalian cells.

Fitzgerald DJ, Burgoyne RD, Haynes LP - Biochim. Biophys. Acta (2007)

Bottom Line: Published in vitro evidence has suggested that other members of the calmodulin super-family, in particular the neuronal calcium sensor (NCS) proteins, can similarly modulate calcineurin activity.In an extension to these analyses we have also examined the effects of over-expression of NCS-1 or NCS-1 mutants on calcineurin signalling in HeLa cells in experiments examining the dephosphorylation of an NFAT-GFP reporter construct as a readout of calcineurin activity.Results from these experiments indicate that NCS-1 was not able to detectably modulate calcineurin/NFAT signalling in a live mammalian cell system, findings that are consistent with the idea that calmodulin and not NCS-1 or other NCS family proteins is the physiologically relevant modulator of calcineurin activity.

View Article: PubMed Central - PubMed

Affiliation: The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool, L69 3BX, UK.

ABSTRACT
Calcium activated gene transcription through Nuclear Factor of Activated T-cells, (NFAT) proteins, is emerging as a ubiquitous mechanism for the control of important physiological processes. Of the five mammalian NFAT isoforms, transcriptional activities of NFATs 1-4 are stimulated by a calcium driven association between the ubiquitous phosphatase calcineurin and the calcium-sensing protein calmodulin. Published in vitro evidence has suggested that other members of the calmodulin super-family, in particular the neuronal calcium sensor (NCS) proteins, can similarly modulate calcineurin activity. In this study we have assessed the ability of NCS proteins to interact directly with calcineurin in vitro and report a specific if weak association between various NCS proteins and the phosphatase. In an extension to these analyses we have also examined the effects of over-expression of NCS-1 or NCS-1 mutants on calcineurin signalling in HeLa cells in experiments examining the dephosphorylation of an NFAT-GFP reporter construct as a readout of calcineurin activity. Results from these experiments indicate that NCS-1 was not able to detectably modulate calcineurin/NFAT signalling in a live mammalian cell system, findings that are consistent with the idea that calmodulin and not NCS-1 or other NCS family proteins is the physiologically relevant modulator of calcineurin activity.

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NCS-1 and NCS-1 mutant proteins do not affect dephosphorylation of NFAT-GFP over an acute time course of intracellular Ca2+ elevation. (a) Anti-GFP western blots on HeLa cell lysates from cells transfected with NFAT-GFP and the indicated pcDNA based expression constructs. Cells were treated with Ca2+/ionomycin for the indicated times and then lysed directly into SDS dissociation buffer, separated on SDS-PAGE (12.5% gel) and transferred to nitrocellulose for western blotting with an anti-GFP antibody. Fully phosphorylated NFAT-GFP (P, upper arrow) present at time 0 for all transfection conditions was dephosphorylated on Ca2+/ionomycin treatment as monitored by the appearance of more rapidly migrating species by the 5 min time point for all transfection conditions (De-P, lower arrow). (b) Anti-calcineurin western blot on samples derived from an in vitro binding assay where recombinant CN (1 μM) was incubated with GST control protein, GST-NCS-1 or GST-NCS-1E120Q (all at 1 μM) in the absence (-Ca2+) or presence (+Ca2+) of 1 μM free Ca2+. Samples from duplicate incubations are presented.
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fig3: NCS-1 and NCS-1 mutant proteins do not affect dephosphorylation of NFAT-GFP over an acute time course of intracellular Ca2+ elevation. (a) Anti-GFP western blots on HeLa cell lysates from cells transfected with NFAT-GFP and the indicated pcDNA based expression constructs. Cells were treated with Ca2+/ionomycin for the indicated times and then lysed directly into SDS dissociation buffer, separated on SDS-PAGE (12.5% gel) and transferred to nitrocellulose for western blotting with an anti-GFP antibody. Fully phosphorylated NFAT-GFP (P, upper arrow) present at time 0 for all transfection conditions was dephosphorylated on Ca2+/ionomycin treatment as monitored by the appearance of more rapidly migrating species by the 5 min time point for all transfection conditions (De-P, lower arrow). (b) Anti-calcineurin western blot on samples derived from an in vitro binding assay where recombinant CN (1 μM) was incubated with GST control protein, GST-NCS-1 or GST-NCS-1E120Q (all at 1 μM) in the absence (-Ca2+) or presence (+Ca2+) of 1 μM free Ca2+. Samples from duplicate incubations are presented.

Mentions: Our analyses suggested that, over a 7 minute time course, none of the NCS-1 constructs under examination were capable of affecting the dephosphorylation of NFAT-GFP in response to [Ca2+]i elevation and, by inference, nor were they able to influence CN activity in intact mammalian cells. These whole cell data were consistent with our in vitro binding studies however we decided to complete our investigation with two further analyses, firstly examining whether or not NCS-1 might exert a far more subtle effect on the CN/NFAT pathway over an acute time course of [Ca2+]i elevation (Fig. 3a). In these experiments, HeLa cells were again transfected with NFAT-GFP along with control or NCS-1 expression constructs however dephosphorylation was monitored over a short 5 minute time course. For all constructs tested NFAT dephosphorylation was clearly apparent by the 5 minute time point (a 25 minute time point was also included to demonstrate complete dephosphorylation of NFAT-GFP) as determined by gel shift of the NFAT-GFP. Analysis of these earlier time points indicated there was no discernable difference in the pattern of dephosphorylation between any of the NCS-1 constructs tested and control transfected cells.


Neuronal calcium sensor proteins are unable to modulate NFAT activation in mammalian cells.

Fitzgerald DJ, Burgoyne RD, Haynes LP - Biochim. Biophys. Acta (2007)

NCS-1 and NCS-1 mutant proteins do not affect dephosphorylation of NFAT-GFP over an acute time course of intracellular Ca2+ elevation. (a) Anti-GFP western blots on HeLa cell lysates from cells transfected with NFAT-GFP and the indicated pcDNA based expression constructs. Cells were treated with Ca2+/ionomycin for the indicated times and then lysed directly into SDS dissociation buffer, separated on SDS-PAGE (12.5% gel) and transferred to nitrocellulose for western blotting with an anti-GFP antibody. Fully phosphorylated NFAT-GFP (P, upper arrow) present at time 0 for all transfection conditions was dephosphorylated on Ca2+/ionomycin treatment as monitored by the appearance of more rapidly migrating species by the 5 min time point for all transfection conditions (De-P, lower arrow). (b) Anti-calcineurin western blot on samples derived from an in vitro binding assay where recombinant CN (1 μM) was incubated with GST control protein, GST-NCS-1 or GST-NCS-1E120Q (all at 1 μM) in the absence (-Ca2+) or presence (+Ca2+) of 1 μM free Ca2+. Samples from duplicate incubations are presented.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2258317&req=5

fig3: NCS-1 and NCS-1 mutant proteins do not affect dephosphorylation of NFAT-GFP over an acute time course of intracellular Ca2+ elevation. (a) Anti-GFP western blots on HeLa cell lysates from cells transfected with NFAT-GFP and the indicated pcDNA based expression constructs. Cells were treated with Ca2+/ionomycin for the indicated times and then lysed directly into SDS dissociation buffer, separated on SDS-PAGE (12.5% gel) and transferred to nitrocellulose for western blotting with an anti-GFP antibody. Fully phosphorylated NFAT-GFP (P, upper arrow) present at time 0 for all transfection conditions was dephosphorylated on Ca2+/ionomycin treatment as monitored by the appearance of more rapidly migrating species by the 5 min time point for all transfection conditions (De-P, lower arrow). (b) Anti-calcineurin western blot on samples derived from an in vitro binding assay where recombinant CN (1 μM) was incubated with GST control protein, GST-NCS-1 or GST-NCS-1E120Q (all at 1 μM) in the absence (-Ca2+) or presence (+Ca2+) of 1 μM free Ca2+. Samples from duplicate incubations are presented.
Mentions: Our analyses suggested that, over a 7 minute time course, none of the NCS-1 constructs under examination were capable of affecting the dephosphorylation of NFAT-GFP in response to [Ca2+]i elevation and, by inference, nor were they able to influence CN activity in intact mammalian cells. These whole cell data were consistent with our in vitro binding studies however we decided to complete our investigation with two further analyses, firstly examining whether or not NCS-1 might exert a far more subtle effect on the CN/NFAT pathway over an acute time course of [Ca2+]i elevation (Fig. 3a). In these experiments, HeLa cells were again transfected with NFAT-GFP along with control or NCS-1 expression constructs however dephosphorylation was monitored over a short 5 minute time course. For all constructs tested NFAT dephosphorylation was clearly apparent by the 5 minute time point (a 25 minute time point was also included to demonstrate complete dephosphorylation of NFAT-GFP) as determined by gel shift of the NFAT-GFP. Analysis of these earlier time points indicated there was no discernable difference in the pattern of dephosphorylation between any of the NCS-1 constructs tested and control transfected cells.

Bottom Line: Published in vitro evidence has suggested that other members of the calmodulin super-family, in particular the neuronal calcium sensor (NCS) proteins, can similarly modulate calcineurin activity.In an extension to these analyses we have also examined the effects of over-expression of NCS-1 or NCS-1 mutants on calcineurin signalling in HeLa cells in experiments examining the dephosphorylation of an NFAT-GFP reporter construct as a readout of calcineurin activity.Results from these experiments indicate that NCS-1 was not able to detectably modulate calcineurin/NFAT signalling in a live mammalian cell system, findings that are consistent with the idea that calmodulin and not NCS-1 or other NCS family proteins is the physiologically relevant modulator of calcineurin activity.

View Article: PubMed Central - PubMed

Affiliation: The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool, L69 3BX, UK.

ABSTRACT
Calcium activated gene transcription through Nuclear Factor of Activated T-cells, (NFAT) proteins, is emerging as a ubiquitous mechanism for the control of important physiological processes. Of the five mammalian NFAT isoforms, transcriptional activities of NFATs 1-4 are stimulated by a calcium driven association between the ubiquitous phosphatase calcineurin and the calcium-sensing protein calmodulin. Published in vitro evidence has suggested that other members of the calmodulin super-family, in particular the neuronal calcium sensor (NCS) proteins, can similarly modulate calcineurin activity. In this study we have assessed the ability of NCS proteins to interact directly with calcineurin in vitro and report a specific if weak association between various NCS proteins and the phosphatase. In an extension to these analyses we have also examined the effects of over-expression of NCS-1 or NCS-1 mutants on calcineurin signalling in HeLa cells in experiments examining the dephosphorylation of an NFAT-GFP reporter construct as a readout of calcineurin activity. Results from these experiments indicate that NCS-1 was not able to detectably modulate calcineurin/NFAT signalling in a live mammalian cell system, findings that are consistent with the idea that calmodulin and not NCS-1 or other NCS family proteins is the physiologically relevant modulator of calcineurin activity.

Show MeSH
Related in: MedlinePlus