A Doubletime Nuclear Localization Signal Mediates an Interaction with Bride of Doubletime to Promote Circadian Function. (August 2015)

• Record Type: Journal Article
• Authors: Venkatesan, Anandakrishnan; Fan, Jin-Yuan; Nauman, Christopher; Price, Jeffrey L.
• Is Part Of: Journal of biological rhythms. Volume 30:Number 4(2015:Aug.)
• Page Start: 302
• Page End: 317
• Publication Date: 2015-08
• Subjects: DBTK/R -- PER phosphorylation -- circadian rhythm -- Drosophila clock -- protein degradation; Biological rhythms -- Periodicals; Circadian rhythms -- Periodicals; 571.77
• DOI: 10.1177/0748730415588189
• Languages: English
• ISSNs: 0748-7304

Abstract

Doubletime (DBT) has an essential circadian role in Drosophila melanogaster because it phosphorylates Period (PER). To determine if DBT antagonism can produce distinct effects in the cytosol and nucleus, forms of a dominant negative DBT K/R with these 2 alternative localizations were produced. DBT has a putative nuclear localization signal (NLS), and mutation of this signal confers cytosolic localization of DBT in the lateral neurons of Drosophila clock cells in the brain. By contrast, addition of a strong NLS domain (e.g., SV40 NLS) to DBT's C terminus leads to more nuclear localization. Expression of DBT K/R with the mutated NLS (DBT K/R NLS − ) using a tim GAL4 driver does not alter the circadian period of locomotor activity, and the daily oscillations of PER detected by immunoblot and immunofluorescence persist, like those of wild-type flies. By contrast, expression of DBT K/R with the strong NLS (DBT K/R stNLS) using the tim GAL4 driver lengthens period more strongly than DBT K/R, with damped oscillations of PER phosphorylation and localization. Both DBT K/R and DBT WT without the NLS fail to interact with Bride of Doubletime (BDBT) protein, which is related to FK506-binding proteins and shown to interact with DBT to enhance its circadian function. This result suggests that the DBT K/R NLS − has lost its dominant negative property because it does not form normal clock protein complexes. DBT WT proteins with the same changes (NLS − and stNLS) also produce equivalent changes in localization that do not produce opposite period phenotypes. Additionally, a DBT K/R protein with both the stNLS and NLS − mutation does not affect circadian period, although it is nuclear, demonstrating that the lack of a dominant negative for the DBT K/R NLS − is not due to failure to localize to nuclei. Finally, bdbt RNAi increases the cytosolic localization of DBT K/R but not of DBT WT, suggesting a role for BDBT in DBT kinase-dependent nuclear localization of DBT.