Limits...
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.

Show MeSH
CK2αTik Circadian Overexpression Alters PER Protein Levels and Mobility(A) Representative PER western blots demonstrating differences in PER protein amount and mobility with UASTik overexpression during constant conditions. (a) y w, (b) timGal4/+; UASTikR/+, (c) Tik/+, (d) timGal4/+; UASTikT1/+, and (e) timGal4; UASTikT1.(B) Quantification of PER protein levels indicate that decreasing CK2α function results in elevated levels and delayed decline of PER. +/+: y w, timTik: timGal4/+; UASTikT1/+; timTik2x: timGal4; UASTikT1. CT: circadian time. Quantification done from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211540&req=5

pgen-0040012-g005: CK2αTik Circadian Overexpression Alters PER Protein Levels and Mobility(A) Representative PER western blots demonstrating differences in PER protein amount and mobility with UASTik overexpression during constant conditions. (a) y w, (b) timGal4/+; UASTikR/+, (c) Tik/+, (d) timGal4/+; UASTikT1/+, and (e) timGal4; UASTikT1.(B) Quantification of PER protein levels indicate that decreasing CK2α function results in elevated levels and delayed decline of PER. +/+: y w, timTik: timGal4/+; UASTikT1/+; timTik2x: timGal4; UASTikT1. CT: circadian time. Quantification done from three independent experiments.

Mentions: To quantitatively examine the effect of CK2αTik on PER cycling and phosphorylation, we used western blots of whole head extracts on the first day of DD. The far majority of PER in whole heads is expressed in the eye [45]. To examine CK2αTik effects we used the timGAL4 driver that includes strong expression in the eye. In wild-type flies, PER phosphorylation (evident as reduced mobility) peaks in the early subjective morning (CT1, Figure 5Aa). PER levels are subsequently reduced, presumably reflecting phosphorylation-induced degradation. PER begins to appear early in the subjective night (CT13, Figure 5Aa), and levels accumulate during the night as PER becomes progressively more phosphorylated. In Tik/+ and timTik flies, both level and mobility rhythms are delayed, consistent with a lengthened period in these flies (Figure 5Ac, 5Ad, and 5B), while expression of UASTikR was not detectably different than wild type (Figure 5Ab).


Dominant-negative CK2alpha induces potent effects on circadian rhythmicity.

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

CK2αTik Circadian Overexpression Alters PER Protein Levels and Mobility(A) Representative PER western blots demonstrating differences in PER protein amount and mobility with UASTik overexpression during constant conditions. (a) y w, (b) timGal4/+; UASTikR/+, (c) Tik/+, (d) timGal4/+; UASTikT1/+, and (e) timGal4; UASTikT1.(B) Quantification of PER protein levels indicate that decreasing CK2α function results in elevated levels and delayed decline of PER. +/+: y w, timTik: timGal4/+; UASTikT1/+; timTik2x: timGal4; UASTikT1. CT: circadian time. Quantification done from three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-0040012-g005: CK2αTik Circadian Overexpression Alters PER Protein Levels and Mobility(A) Representative PER western blots demonstrating differences in PER protein amount and mobility with UASTik overexpression during constant conditions. (a) y w, (b) timGal4/+; UASTikR/+, (c) Tik/+, (d) timGal4/+; UASTikT1/+, and (e) timGal4; UASTikT1.(B) Quantification of PER protein levels indicate that decreasing CK2α function results in elevated levels and delayed decline of PER. +/+: y w, timTik: timGal4/+; UASTikT1/+; timTik2x: timGal4; UASTikT1. CT: circadian time. Quantification done from three independent experiments.
Mentions: To quantitatively examine the effect of CK2αTik on PER cycling and phosphorylation, we used western blots of whole head extracts on the first day of DD. The far majority of PER in whole heads is expressed in the eye [45]. To examine CK2αTik effects we used the timGAL4 driver that includes strong expression in the eye. In wild-type flies, PER phosphorylation (evident as reduced mobility) peaks in the early subjective morning (CT1, Figure 5Aa). PER levels are subsequently reduced, presumably reflecting phosphorylation-induced degradation. PER begins to appear early in the subjective night (CT13, Figure 5Aa), and levels accumulate during the night as PER becomes progressively more phosphorylated. In Tik/+ and timTik flies, both level and mobility rhythms are delayed, consistent with a lengthened period in these flies (Figure 5Ac, 5Ad, and 5B), while expression of UASTikR was not detectably different than wild type (Figure 5Ab).

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