The Drosophila gene shaggy (sgg) has recently been shown to play a role in the fly circadian oscillator. SGG is the Drosophila ortholog of glycogen synthase kinase-3 (GSK-3), and appears to affect nuclear entry of protein complexes made up of the PERIOD (PER) and TIMELESS (TIM) proteins by promoting the phosphorylation of TIM. Since sgg loss-of-function mutations result in lethality, however, it has been difficult to determine what effect the loss of sgg function would have on the circadian clock. The work proposed here is aimed at further elucidating the role of SGG in the Drosophila circadian clock, as well as investigating whether GSK-3 plays a comparable role in mammals. In the fly, the focus of this proposal is to determine what effect the loss of SGG/GSK-3 phosphorylation of TIM would have on the circadian clock. Putative GSK-3 phosphorylation sites within TIM will be identified, mutated to residues that cannot be phosphorylated, and then placed into tim genomic transgenes to create transgenic flies. The mutant transgenic flies will be used to determine whether the phosphorylation site mutations indeed affect the phosphorylation and light responsiveness of TIM in vivo, and also whether the mutations impede the nuclear translocation of PER/TIM complexes. In the mammalian system, gsk-3 expression levels will be manipulated in mammalian fibroblast cells. The cells will be monitored for defects in circadian rhythmicity to assess a possible role for GSK-3 in the mammalian clock. In vitro kinase assays will be used to identify potential substrates of GSK-3 in the mammalian clock, and regulation of GSK-3 activity will be examined. Additionally, an involvement of GSK-3 in the mammalian clock may have important implications for the understanding and treatment of bipolar disorder in humans.
