Kinetics of doubletime kinase-dependent degradation of the Drosophila period protein

Abstract

Robust circadian oscillations of the proteins PERIOD (PER) and TIMELESS (TIM) are hallmarks of a functional clock in the fruit fly Drosophila melanogaster. Early morning phosphorylation of PER by the kinase Doubletime (DBT) and subsequent PER turnover is an essential step in the functioning of the Drosophila circadian clock. Here using time-lapse fluorescence microscopy we study PER stability in the presence of DBT and its short, long, arrhythmic, and inactive mutants in S2 cells. We observe robust PER degradation in a DBT allele-specific manner. With the exception of doubletime-short (DBT S), all mutants produce differential PER degradation profiles that show direct correspondence with their respective Drosophila behavioral phenotypes. The kinetics of PER degradation with DBT S in cell culture resembles that with wild-type DBT and posits that, in flies DBT S likely does not modulate the clock by simply affecting PER degradation kinetics. For all the other tested DBT alleles, the study provides a simple model in which the changes in Drosophila behavioral rhythms can be explained solely by changes in the rate of PER degradation. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.