Sekine Tatsumori, Yamaguchi Terumi, Hamano Kunikatsu, Young Michael W, Shimoda Masami, Saez Lino
Division of Insect Sciences, National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba, Ibaraki 305-8634, Japan.
J Biol Rhythms. 2008 Feb;23(1):3-15. doi: 10.1177/0748730407311652.
Double-time (dbt) is a casein kinase gene involved in cell survival, proliferation, and circadian rhythms in the fruit fly, Drosophila melanogaster. Genetic and biochemical studies have shown that dbt and its mammalian ortholog casein kinase I epsilon (hckI epsilon) regulate the circadian phosphorylation of period (per), thus controlling per subcellular localization and stability. Mutations in these kinases can shorten the circadian period in both mammals and Drosophila. Since similar activities in circadian clock have been described for these kinases, we investigated whether the expression of mammalian casein kinase I can replace the activity of dbt in flies. Global expression of the full-length dbt rescued lethality of the null mutant dbt revVIII and rescued flies showed normal locomotor activity rhythms. Global expression of dbt also restored the locomotor activity rhythm of the arrhythmic genotype, dbt ar/dbt revVIII. In contrast, global expression of hckI epsilon or hckI alpha did not rescue lethality or locomotor activity of dbt mutants. Furthermore dbt overexpression in wild-type clock cells had only a small effect on period length, whereas hckI epsilon expression in clock cells greatly lengthened period to ~30.5 hours and increased the number of arrhythmic flies. These results indicate that hckI epsilon cannot replace the activity of dbt in flies despite the high degree of similarity in primary sequence and kinase function. Moreover, expression of hck Iepsilon in flies appears to interfere with dbt activity. Thus, caution should be used in interpreting assays that measure activity of mammalian casein kinase mutants in Drosophila, or that employ vertebrate CKI in studies of dPER phosphorylations.
双时（dbt）是一种酪蛋白激酶基因，参与果蝇（黑腹果蝇）的细胞存活、增殖和昼夜节律。遗传学和生物化学研究表明，dbt及其哺乳动物直系同源基因酪蛋白激酶Iε（hckIε）调节周期蛋白（per）的昼夜磷酸化，从而控制per的亚细胞定位和稳定性。这些激酶的突变可缩短哺乳动物和果蝇的昼夜周期。由于已报道这些激酶在生物钟中具有相似活性，我们研究了哺乳动物酪蛋白激酶I的表达是否能替代果蝇中dbt的活性。全长dbt的整体表达挽救了无效突变体dbt revVIII的致死性，挽救后的果蝇表现出正常的运动活动节律。dbt的整体表达还恢复了无节律基因型dbt ar/dbt revVIII的运动活动节律。相比之下，hckIε或hckIα的整体表达未能挽救dbt突变体的致死性或运动活动。此外，在野生型生物钟细胞中过表达dbt对周期长度只有很小的影响，而在生物钟细胞中表达hckIε则使周期大大延长至约30.5小时，并增加了无节律果蝇的数量。这些结果表明，尽管hckIε与dbt在一级序列和激酶功能上高度相似，但它不能替代果蝇中dbt的活性。此外，在果蝇中表达hckIε似乎会干扰dbt的活性。因此，在解释测量果蝇中哺乳动物酪蛋白激酶突变体活性的试验或在研究dPER磷酸化时使用脊椎动物CKI的试验结果时应谨慎。
