Nawathean Pipat, Rosbash Michael
Howard Hughes Medical Institute, Biology Department MS 008, Brandeis University, 415 South Street, Waltham, MA 02454, USA.
Mol Cell. 2004 Jan 30;13(2):213-23. doi: 10.1016/s1097-2765(03)00503-3.
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.
在所有经过基因研究的模式生物中，基因表达的负反馈回路有助于昼夜节律机制。在果蝇系统中，有人提出时钟蛋白合成与功能之间的延迟是由于磷酸化调节的核进入。为了验证这一假设，我们在培养的S2细胞中检测了PER磷酸化、核定位和转录抑制活性之间的关系。结果表明，两种假定的PER激酶DBT和CKII共同作用使PER磷酸化并增加抑制活性。将激酶抑制与PER核输出抑制相结合的实验表明，磷酸化直接影响PER抑制活性，并且PER核定位是活性PER与DNA或染色质结合的间接结果。这一解释进一步表明，果蝇中PER核定位的昼夜调节反映了PER转录活性的变化，而不是PER核输入或输出活性的变化。
