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Dominant-negative CK2alpha induces potent effects on circadian rhythmicity.

Smith EM, Lin JM, Meissner RA, Allada R - PLoS Genet. (2007)

Bottom Line: CK2alpha(Tik), when targeted to a subset of pacemaker neurons, generates period splitting, resulting in flies exhibiting both long and near 24-h periods.These behavioral effects are evident even when CK2alpha(Tik) expression is induced only during adulthood, implicating an acute role for CK2alpha function in circadian rhythms.CK2alpha(Tik) expression results in reduced PER phosphorylation, delayed nuclear entry, and dampened cycling with elevated trough levels of PER.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois, United States of America.

ABSTRACT
Circadian clocks organize the precise timing of cellular and behavioral events. In Drosophila, circadian clocks consist of negative feedback loops in which the clock component PERIOD (PER) represses its own transcription. PER phosphorylation is a critical step in timing the onset and termination of this feedback. The protein kinase CK2 has been linked to circadian timing, but the importance of this contribution is unclear; it is not certain where and when CK2 acts to regulate circadian rhythms. To determine its temporal and spatial functions, a dominant negative mutant of the catalytic alpha subunit, CK2alpha(Tik), was targeted to circadian neurons. Behaviorally, CK2alpha(Tik) induces severe period lengthening (approximately 33 h), greater than nearly all known circadian mutant alleles, and abolishes detectable free-running behavioral rhythmicity at high levels of expression. CK2alpha(Tik), when targeted to a subset of pacemaker neurons, generates period splitting, resulting in flies exhibiting both long and near 24-h periods. These behavioral effects are evident even when CK2alpha(Tik) expression is induced only during adulthood, implicating an acute role for CK2alpha function in circadian rhythms. CK2alpha(Tik) expression results in reduced PER phosphorylation, delayed nuclear entry, and dampened cycling with elevated trough levels of PER. Heightened trough levels of per transcript accompany increased protein levels, suggesting that CK2alpha(Tik) disturbs negative feedback of PER on its own transcription. Taken together, these in vivo data implicate a central role of CK2alpha function in timing PER negative feedback in adult circadian neurons.

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Circadian CK2α Loss of Function Impacts Clock Gene RNA Levels(A) Overexpression of one copy of UASTik leads to elevated levels of per transcript during early subjective night, while further diminishing CK2α function with two copies each of timGal4 and UASTik greatly diminishes the amplitude of per RNA cycling.(B) Analysis of vri transcript reveals a similar pattern of delayed and dampened transcript persistence when CK2α activity is inhibited in circadian cells. +/+: y w, timTik: timGal4/+; UASTikT1/+; timTik2x: timGal4; UASTikT1. CT: circadian time. Values obtained from two to three independent experiments.
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pgen-0040012-g006: Circadian CK2α Loss of Function Impacts Clock Gene RNA Levels(A) Overexpression of one copy of UASTik leads to elevated levels of per transcript during early subjective night, while further diminishing CK2α function with two copies each of timGal4 and UASTik greatly diminishes the amplitude of per RNA cycling.(B) Analysis of vri transcript reveals a similar pattern of delayed and dampened transcript persistence when CK2α activity is inhibited in circadian cells. +/+: y w, timTik: timGal4/+; UASTikT1/+; timTik2x: timGal4; UASTikT1. CT: circadian time. Values obtained from two to three independent experiments.

Mentions: Previous studies have implicated CK2 in promoting PER repression of CLK activation [22]. However, these studies were performed in cultured Drosophila S2 cells which do not harbor functioning circadian clocks. To test the hypothesis that CK2 promotes PER repression in vivo, we examined circadian transcription in UASTik expressing flies. Levels of two CLK-activated transcripts, per and vrille (vri) [46] were analyzed using quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR). We hypothesized that if negative feedback is unaffected in UASTik expressing flies, then elevated PER levels would strongly repress CLK, reducing per and vri transcript levels. If negative feedback is disrupted, then elevated PER levels would fail to appropriately repress per and vri transcription. Expression of dominant-negative UASTik in circadian neurons in timTik flies postpones the decline in per transcript until early subjective night (Figure 6A), consistent with the effect of CK2α loss of function in the heterozygous Tik/+ mutant. Whereas wild type per transcription peaks around CT9–13, per levels do not achieve maximum until CT13–17 in timTik flies, and a similar pattern emerges from analysis of the vri transcript (Figure 6B).


Dominant-negative CK2alpha induces potent effects on circadian rhythmicity.

Smith EM, Lin JM, Meissner RA, Allada R - PLoS Genet. (2007)

Circadian CK2α Loss of Function Impacts Clock Gene RNA Levels(A) Overexpression of one copy of UASTik leads to elevated levels of per transcript during early subjective night, while further diminishing CK2α function with two copies each of timGal4 and UASTik greatly diminishes the amplitude of per RNA cycling.(B) Analysis of vri transcript reveals a similar pattern of delayed and dampened transcript persistence when CK2α activity is inhibited in circadian cells. +/+: y w, timTik: timGal4/+; UASTikT1/+; timTik2x: timGal4; UASTikT1. CT: circadian time. Values obtained from two to three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211540&req=5

pgen-0040012-g006: Circadian CK2α Loss of Function Impacts Clock Gene RNA Levels(A) Overexpression of one copy of UASTik leads to elevated levels of per transcript during early subjective night, while further diminishing CK2α function with two copies each of timGal4 and UASTik greatly diminishes the amplitude of per RNA cycling.(B) Analysis of vri transcript reveals a similar pattern of delayed and dampened transcript persistence when CK2α activity is inhibited in circadian cells. +/+: y w, timTik: timGal4/+; UASTikT1/+; timTik2x: timGal4; UASTikT1. CT: circadian time. Values obtained from two to three independent experiments.
Mentions: Previous studies have implicated CK2 in promoting PER repression of CLK activation [22]. However, these studies were performed in cultured Drosophila S2 cells which do not harbor functioning circadian clocks. To test the hypothesis that CK2 promotes PER repression in vivo, we examined circadian transcription in UASTik expressing flies. Levels of two CLK-activated transcripts, per and vrille (vri) [46] were analyzed using quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR). We hypothesized that if negative feedback is unaffected in UASTik expressing flies, then elevated PER levels would strongly repress CLK, reducing per and vri transcript levels. If negative feedback is disrupted, then elevated PER levels would fail to appropriately repress per and vri transcription. Expression of dominant-negative UASTik in circadian neurons in timTik flies postpones the decline in per transcript until early subjective night (Figure 6A), consistent with the effect of CK2α loss of function in the heterozygous Tik/+ mutant. Whereas wild type per transcription peaks around CT9–13, per levels do not achieve maximum until CT13–17 in timTik flies, and a similar pattern emerges from analysis of the vri transcript (Figure 6B).

Bottom Line: CK2alpha(Tik), when targeted to a subset of pacemaker neurons, generates period splitting, resulting in flies exhibiting both long and near 24-h periods.These behavioral effects are evident even when CK2alpha(Tik) expression is induced only during adulthood, implicating an acute role for CK2alpha function in circadian rhythms.CK2alpha(Tik) expression results in reduced PER phosphorylation, delayed nuclear entry, and dampened cycling with elevated trough levels of PER.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois, United States of America.

ABSTRACT
Circadian clocks organize the precise timing of cellular and behavioral events. In Drosophila, circadian clocks consist of negative feedback loops in which the clock component PERIOD (PER) represses its own transcription. PER phosphorylation is a critical step in timing the onset and termination of this feedback. The protein kinase CK2 has been linked to circadian timing, but the importance of this contribution is unclear; it is not certain where and when CK2 acts to regulate circadian rhythms. To determine its temporal and spatial functions, a dominant negative mutant of the catalytic alpha subunit, CK2alpha(Tik), was targeted to circadian neurons. Behaviorally, CK2alpha(Tik) induces severe period lengthening (approximately 33 h), greater than nearly all known circadian mutant alleles, and abolishes detectable free-running behavioral rhythmicity at high levels of expression. CK2alpha(Tik), when targeted to a subset of pacemaker neurons, generates period splitting, resulting in flies exhibiting both long and near 24-h periods. These behavioral effects are evident even when CK2alpha(Tik) expression is induced only during adulthood, implicating an acute role for CK2alpha function in circadian rhythms. CK2alpha(Tik) expression results in reduced PER phosphorylation, delayed nuclear entry, and dampened cycling with elevated trough levels of PER. Heightened trough levels of per transcript accompany increased protein levels, suggesting that CK2alpha(Tik) disturbs negative feedback of PER on its own transcription. Taken together, these in vivo data implicate a central role of CK2alpha function in timing PER negative feedback in adult circadian neurons.

Show MeSH
Related in: MedlinePlus