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

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.