Characterization of Pdp1ε in drosophila circadian clock

Abstract

Circadian rhythms of various physiological events are driven by endogenous molecular clocks. Two transcriptional feedback loops maintain molecular clock system in Drosophila melanogaster. dCLOCK (dCLK) and CYCLE (CYC) form a heterodimer, and activates period (per) and timeless (tim) transcription via binding to specific enhancer element (E box). The PER and TIM proteins accumulate and translocate to nucleus, and repress the transcriptional activity of dCLK/CYC. VRILLE (VRI) and PAR DOMAIN PROTEIN 1ε (PDP1ε) which are induced by dCLK/CYC feedback to regulate clk expression as a transcriptional repressor and activator, respectively. Reversible phosphorylation of clock proteins has essential parts of circadian time keeping. Phosphorylation of proteins in circadian system is key steps that regulate the activity, stability and subcellular localization. In this study, we showed that PDP1ε is phosphorylated protein in vivo and in a cultured cell system. DBT kinase phosphorylates PDP1ε in S2 cells and in vitro. In addition, PDP1ε is dephosphorylated by silencing endogenous DBT activity. We also found DBT interacts with PDP1ε. DBT activity of PDP1ε phosphorylation promotes its degradation by ubiquitin-proteasome pathway. PDP1ε is a exclusive nuclear protein. However, PDP1ε nuclear localization was disrupted in absence of DBT activity. These results demonstrate that DBT regulates phosphorylation, stability and localization of PDP1ε, and has multiple targets in circadian timekeeping system. To study the role of Pdp1ε in adult circadian clock, Pdp1-GAL4 and UAS-pdp1 RNAi were generated to inhibit Pdp1ε expression in Pdp1ε expressing neurons. Pdp1 RNAi driven by Pdp1-GAL4 or tim-GAL4 disrupts PDP1ε expression in pacemaker neuron, LNs. Surprisingly, dCLK expression in circadian pacemaker cell of Pdp1ε mutant flies is similar to that in control flies. The core circadian oscillator was not disrupted in Pdp1ε mutants. Other factors may be involved in rgulating dClk mRN...