The DOUBLETIME protein kinase regulates phosphorylation of the Drosophila PDP1ε

Abstract

Reversible phosphorylation of clock proteins plays an important role in circadian timekeeping as it is a key post‐translational mechanism that regulates the activity, stability and subcellular localization of core clock proteins. The kinase DOUBLETIME (DBT), a Drosophila ortholog of mammalian casein kinase Iε, regulates circadian phosphorylation of two essential clock proteins, PERIOD and dCLOCK. We present evidence that Par Domain Protein 1ε (PDP1ε), a transcription factor and mediator of clock output in Drosophila, is phosphorylated in vivo and in cultured cells by DBT activity. We also demonstrate that DBT interacts with PDP1ε and promotes its degradation by the ubiquitin‐proteasome pathway in cultured cells. In addition, PDP1ε nuclear localization is decreased by dbt RNA interference in S2 cell system. These results suggest that DBT regulates phosphorylation, stability and localization of PDP1ε, and that it has multiple targets in the Drosophila circadian system.

The ROP vesicle release factor is required in adult Drosophila glia for normal circadian behavior

“…The high level of PDP1ε observed in UAS-Rop-IR flies suggests that the protein may not be degraded properly. Degradation of PDP1ε is promoted by its own phosphorylation state, and doubletime (DBT or CK1ε) is known to phosphorylate the protein both in vitro and in vivo in a clock-independent manner (Choi et al, 2009 ). Perhaps a slow accumulation of non-degraded PDP1ε in clock neurons—induced by conditional glial knockdown of ROP and altered glia-neuron signaling—leads to the gradual deterioration of rhythmic behavior observed in our studies.…”

High-Amplitude Circadian Rhythms in Drosophila Driven by Calcineurin-Mediated Post-translational Control of sarah

“…Quantification of PER and TIM levels in individual clock neurons was performed as described previously (Lee et al 2017). The primary antibodies used were mouse anti-PIGMENT-DISPERSING FACTOR (PDF) (Development Studies Hybridoma Bank), rabbit anti-PER, and guinea pig anti-TIM (Choi et al 2009). The secondary antibodies used were Alexa Fluor 594-conjugated AffiniPure F(ab')2 fragment goat anti-guinea pig, anti-rabbit, and antimouse, and fluorescein isothiocyanate (FITC)-conjugated AffiniPure F(ab')2 fragment goat anti-rabbit, anti-guinea pig, and anti-mouse (Jackson ImmunoResearch Laboratories).…”