The doubletime and CKII kinases collaborate to potentiate Drosophila PER transcriptional repressor activity.
In all genetically studied model organisms, a negative feedback loop of gene expression contributes to the circadian rhythm mechanism. In the Drosophila system, it has been proposed that the delay between the synthesis and function of clock proteins is due to phosphorylation-regulated nuclear entry. To test this hypothesis, we assayed the relationship between PER phosphorylation, nuclear localization, and transcriptional repression activity in cultured S2 cells. The results indicate that the two putative PER kinases DBT and CKII work together to phosphorylate PER and increase repression activity. Experiments combining kinase inhibition with inhibition of PER nuclear export suggest that phosphorylation directly affects PER repression activity and that PER nuclear localization is an indirect consequence of the association of active PER with DNA or chromatin. This interpretation suggests further that the circadian regulation of PER nuclear localization in flies reflects changes in PER transcriptional activity rather than in PER nuclear import or export activity.
Nawathean
Pipat
P
Howard Hughes Medical Institute, Biology Department MS 008, Brandeis University, 415 South Street, Waltham, MA 02454, USA.
Rosbash
Michael
M
D021581:Active Transport, Cell Nucleus; D000818:Animals; D015153:Blotting, Western; D048149:Casein Kinase 1 epsilon; D047390:Casein Kinase II; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D002940:Circadian Rhythm; D004305:Dose-Response Relationship, Drug; D004330:Drosophila; D029721:Drosophila Proteins / Q000378:metabolism / Q000502:physiology*; D007150:Immunohistochemistry; D008156:Luciferases / Q000378:metabolism; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism*; D056950:Period Circadian Proteins; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology*; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D015533:Transcriptional Activation; D014162:Transfection
D002843:Chromatin; D029721:Drosophila Proteins; D009687:Nuclear Proteins; C539040:PER protein, Drosophila; D056950:Period Circadian Proteins; C113519:dco protein, Drosophila; D008156:Luciferases; D011494:Protein Kinases; D048149:Casein Kinase 1 epsilon; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases