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Functional analysis of the rodent CK1tau mutation in the circadian clock of a marine unicellular alga.

van Ooijen G, Martin SF, Barrios-Llerena ME, Hindle M, Le Bihan T, O'Neill JS, Millar AJ - BMC Cell Biol. (2013)

Bottom Line: Label-free quantitative mass spectrometry of CK1tau overexpressing algae revealed a total of 58 unique phospho-sites that are differentially responsive to CK1tau.Combined with CK1 phosphorylation site prediction tools and previously published wild-type CK1-responsive peptides, this study results in a highly stringent list of upregulated phospho-sites, derived from proteins containing ankyrin repeats, kinase proteins, and phosphoinositide-binding proteins.Proteomic analyses reveal that two thirds of previously reported CK1 overexpression-responsive phospho-sites are shared with CK1tau.

View Article: PubMed Central - HTML - PubMed

Affiliation: SynthSys, University of Edinburgh, Waddington Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JD, UK. Gerben.vanOoijen@ed.ac.uk.

ABSTRACT

Background: Casein Kinase 1 (CK1) is one of few proteins known to affect cellular timekeeping across metazoans, and the naturally occurring CK1tau mutation shortens circadian period in mammals. Functional conservation of a timekeeping function for CK1 in the green lineage was recently identified in the green marine unicell Ostreococcus tauri, in spite of the absence of CK1's transcriptional targets known from other species. The short-period phenotype of CK1tau mutant in mammals depends specifically on increased CK1 activity against PERIOD proteins. To understand how CK1 acts differently upon the algal clock, we analysed the cellular and proteomic effects of CK1tau overexpression in O. tauri.

Results: Overexpression of the CK1tau in O. tauri induces period lengthening identical to overexpression of wild-type CK1, in addition to resistance to CK1 inhibitor IC261. Label-free quantitative mass spectrometry of CK1tau overexpressing algae revealed a total of 58 unique phospho-sites that are differentially responsive to CK1tau. Combined with CK1 phosphorylation site prediction tools and previously published wild-type CK1-responsive peptides, this study results in a highly stringent list of upregulated phospho-sites, derived from proteins containing ankyrin repeats, kinase proteins, and phosphoinositide-binding proteins.

Conclusions: The identical phenotype for overexpression of wild-type CK1 and CK1tau is in line with the absence of critical targets for rodent CK1tau in O. tauri. Proteomic analyses reveal that two thirds of previously reported CK1 overexpression-responsive phospho-sites are shared with CK1tau. These results indicate that the two alleles are functionally indiscriminate in O. tauri, and verify the identified cellular CK1 target proteins in a minimal circadian model organism.

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Overexpression of CK1tau induces long period rhythms. A) Free-running period was analysed in 6 independent transgenic lines overexpressing CK1TAU. Lines were compared against parent line CCA1-LUC in the identical plate position to the transgenic lines. In all cases, a significantly (p < 0.001) long circadian period was observed. B) Examples of traces of overexpression line CK1TAU-OX21 (red) compared to the parent line (black) in free-running conditions. C) Period increase (as in panel A) plotted against overexpression (densitometry of immunoblots, n = 3, as described in the Methods section), showing that in all transgenic lines, overexpression of the tau allele is associated with long period rhythms. D) Subtracted period lengthening of the six CK1tau mutant lines (red bars) compared to the parent line. Combined data of six previously published [18] independent overexpression lines of the wild-type CK1 allele were plotted for comparison (in blue).
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Figure 2: Overexpression of CK1tau induces long period rhythms. A) Free-running period was analysed in 6 independent transgenic lines overexpressing CK1TAU. Lines were compared against parent line CCA1-LUC in the identical plate position to the transgenic lines. In all cases, a significantly (p < 0.001) long circadian period was observed. B) Examples of traces of overexpression line CK1TAU-OX21 (red) compared to the parent line (black) in free-running conditions. C) Period increase (as in panel A) plotted against overexpression (densitometry of immunoblots, n = 3, as described in the Methods section), showing that in all transgenic lines, overexpression of the tau allele is associated with long period rhythms. D) Subtracted period lengthening of the six CK1tau mutant lines (red bars) compared to the parent line. Combined data of six previously published [18] independent overexpression lines of the wild-type CK1 allele were plotted for comparison (in blue).

Mentions: To test the effect of mutating this arginine would be, we made the analogous tau mutation R200C in an overexpression construct of CK1 previously used to prove conserved clock function for CK1 in O. tauri[18]. Similar to the previous study, transgenic lines were generated that overexpressed CK1tau in cells carrying a rhythmically luminescent reporter (CCA1-LUC). After verification of transgene expression, 6 independent CK1tau overexpression lines were selected and compared to the parent line. In all cases, a statistically significant long-period phenotype was observed (Figure 2A,B), associated with an approximately 2-fold overexpression level (Figure 2C). The period lengthening that was observed upon overexpression of CK1tau was in the same range as that resulting from overexpression of the wild-type CK1 allele (Figure 2D) averaged for 6 independent overexpression lines previously published [18]. This result shows that the effects of both alleles on period lengthening are very similar.


Functional analysis of the rodent CK1tau mutation in the circadian clock of a marine unicellular alga.

van Ooijen G, Martin SF, Barrios-Llerena ME, Hindle M, Le Bihan T, O'Neill JS, Millar AJ - BMC Cell Biol. (2013)

Overexpression of CK1tau induces long period rhythms. A) Free-running period was analysed in 6 independent transgenic lines overexpressing CK1TAU. Lines were compared against parent line CCA1-LUC in the identical plate position to the transgenic lines. In all cases, a significantly (p < 0.001) long circadian period was observed. B) Examples of traces of overexpression line CK1TAU-OX21 (red) compared to the parent line (black) in free-running conditions. C) Period increase (as in panel A) plotted against overexpression (densitometry of immunoblots, n = 3, as described in the Methods section), showing that in all transgenic lines, overexpression of the tau allele is associated with long period rhythms. D) Subtracted period lengthening of the six CK1tau mutant lines (red bars) compared to the parent line. Combined data of six previously published [18] independent overexpression lines of the wild-type CK1 allele were plotted for comparison (in blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3852742&req=5

Figure 2: Overexpression of CK1tau induces long period rhythms. A) Free-running period was analysed in 6 independent transgenic lines overexpressing CK1TAU. Lines were compared against parent line CCA1-LUC in the identical plate position to the transgenic lines. In all cases, a significantly (p < 0.001) long circadian period was observed. B) Examples of traces of overexpression line CK1TAU-OX21 (red) compared to the parent line (black) in free-running conditions. C) Period increase (as in panel A) plotted against overexpression (densitometry of immunoblots, n = 3, as described in the Methods section), showing that in all transgenic lines, overexpression of the tau allele is associated with long period rhythms. D) Subtracted period lengthening of the six CK1tau mutant lines (red bars) compared to the parent line. Combined data of six previously published [18] independent overexpression lines of the wild-type CK1 allele were plotted for comparison (in blue).
Mentions: To test the effect of mutating this arginine would be, we made the analogous tau mutation R200C in an overexpression construct of CK1 previously used to prove conserved clock function for CK1 in O. tauri[18]. Similar to the previous study, transgenic lines were generated that overexpressed CK1tau in cells carrying a rhythmically luminescent reporter (CCA1-LUC). After verification of transgene expression, 6 independent CK1tau overexpression lines were selected and compared to the parent line. In all cases, a statistically significant long-period phenotype was observed (Figure 2A,B), associated with an approximately 2-fold overexpression level (Figure 2C). The period lengthening that was observed upon overexpression of CK1tau was in the same range as that resulting from overexpression of the wild-type CK1 allele (Figure 2D) averaged for 6 independent overexpression lines previously published [18]. This result shows that the effects of both alleles on period lengthening are very similar.

Bottom Line: Label-free quantitative mass spectrometry of CK1tau overexpressing algae revealed a total of 58 unique phospho-sites that are differentially responsive to CK1tau.Combined with CK1 phosphorylation site prediction tools and previously published wild-type CK1-responsive peptides, this study results in a highly stringent list of upregulated phospho-sites, derived from proteins containing ankyrin repeats, kinase proteins, and phosphoinositide-binding proteins.Proteomic analyses reveal that two thirds of previously reported CK1 overexpression-responsive phospho-sites are shared with CK1tau.

View Article: PubMed Central - HTML - PubMed

Affiliation: SynthSys, University of Edinburgh, Waddington Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JD, UK. Gerben.vanOoijen@ed.ac.uk.

ABSTRACT

Background: Casein Kinase 1 (CK1) is one of few proteins known to affect cellular timekeeping across metazoans, and the naturally occurring CK1tau mutation shortens circadian period in mammals. Functional conservation of a timekeeping function for CK1 in the green lineage was recently identified in the green marine unicell Ostreococcus tauri, in spite of the absence of CK1's transcriptional targets known from other species. The short-period phenotype of CK1tau mutant in mammals depends specifically on increased CK1 activity against PERIOD proteins. To understand how CK1 acts differently upon the algal clock, we analysed the cellular and proteomic effects of CK1tau overexpression in O. tauri.

Results: Overexpression of the CK1tau in O. tauri induces period lengthening identical to overexpression of wild-type CK1, in addition to resistance to CK1 inhibitor IC261. Label-free quantitative mass spectrometry of CK1tau overexpressing algae revealed a total of 58 unique phospho-sites that are differentially responsive to CK1tau. Combined with CK1 phosphorylation site prediction tools and previously published wild-type CK1-responsive peptides, this study results in a highly stringent list of upregulated phospho-sites, derived from proteins containing ankyrin repeats, kinase proteins, and phosphoinositide-binding proteins.

Conclusions: The identical phenotype for overexpression of wild-type CK1 and CK1tau is in line with the absence of critical targets for rodent CK1tau in O. tauri. Proteomic analyses reveal that two thirds of previously reported CK1 overexpression-responsive phospho-sites are shared with CK1tau. These results indicate that the two alleles are functionally indiscriminate in O. tauri, and verify the identified cellular CK1 target proteins in a minimal circadian model organism.

Show MeSH
Related in: MedlinePlus