Genetic and Biochemical Studies on Protein Phosphorylation in the Circadian Clock of Drosophila Melanogaster
Circadian rhythms in physiology and behavior are observed in almost all phyla. Genetically encoded internal clocks generate such rhythms. Identification of gene products required for the generation and maintenance of endogenous circadian near 24-hr rhythms has led to a paradigm of multiple interlocked transcriptional/translational feedback loops as the basis for molecular circadian oscillators in all studied model systems. Protein phosphorylation plays an essential role, regulating the stability, activity and subcellular localization of proteins that constitute the biological clock. This study investigates the role of the protein kinase Doubletime, a Drosophila ortholog of casein kinase Is, in the fruit fly circadian clock. For the first time enzymatically active Doubletime protein is produced and direct phosphorylation of clock protein Period is demonstrated. Phosphorylation sites are identified and their functional significance is tested in a cell culture system. An in vivo analysis of a Period mutant that eliminates one of the identified phosphorylation sites is also carried out. The analysis suggests that phosphorylation dependent regulation of Period protein stability. Transcriptional repressor activity and possibly subcellular localization may all be regulated in an integrated fashion that involves two sequence motifs in the center of the Period protein with high affinity for phosphorylation by Doubletime.
circadian clocks, circadian rhythms, drosophila melanogaster, Life Sciences
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