title: A Doubletime Nuclear Localization Signal Mediates an Interaction with Bride of Doubletime to Promote Circadian Function.
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 timGAL4 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 timGAL4 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.
authors.affiliation: School of Biological Sciences, Division of Molecular Biology and Biochemistry, University of Missouri-Kansas City, Kansas City, Missouri Department of Physiology, The University of Texas Health Science Center at San Antonio STRF-Greehey North Campus, San Antonio, Texas.
authors.forename: Anandakrishnan
authors.initials: A
authors.lastname: Venkatesan
authors.affiliation: School of Biological Sciences, Division of Molecular Biology and Biochemistry, University of Missouri-Kansas City, Kansas City, Missouri.
authors.forename: Jin-Yuan
authors.initials: JY
authors.lastname: Fan
authors.affiliation: School of Biological Sciences, Division of Molecular Biology and Biochemistry, University of Missouri-Kansas City, Kansas City, Missouri.
authors.forename: Christopher
authors.initials: C
authors.lastname: Nauman
authors.affiliation: School of Biological Sciences, Division of Molecular Biology and Biochemistry, University of Missouri-Kansas City, Kansas City, Missouri School of Medicine, Department of Neurology and Cognitive Neuroscience, University of Missouri-Kansas City, Kansas City, Missouri pricejL@umkc.edu.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Price
mesh_terms: D000818:Animals; D001921:Brain / Q000166:cytology; D056926:CLOCK Proteins / Q000235:genetics / Q000378:metabolism; D048149:Casein Kinase 1 epsilon / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002940:Circadian Rhythm; D003600:Cytosol / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000166:cytology / Q000235:genetics* / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism*; D019913:Nuclear Localization Signals / Q000235:genetics / Q000502:physiology*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D056950:Period Circadian Proteins / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation; D034622:RNA Interference; D022021:Tacrolimus Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C000607166:BDBT protein, Drosophila; C120526:Clk protein, Drosophila; D029721:Drosophila Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D056950:Period Circadian Proteins; C113519:dco protein, Drosophila; D056926:CLOCK Proteins; D048149:Casein Kinase 1 epsilon; D022021:Tacrolimus Binding Proteins
keywords: DBTK/R; Drosophila clock; PER phosphorylation; circadian rhythm; protein degradation